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A. 
rticulture. 

:  SYSTEM. 


5  Centimeters  divided  into 
%  Centimeters. 

{visions  and  multiples. 

•;  Millimeter  (m.m.),  i-ioooths  Meter;   Microti 
i  Micron  is  the  unit  in  Micrometry  (§  157). 
neasuring  roads  and  other  long  distances. 

1 

irdinary  masses,  like  groceries,  etc. 

Liter.      This  is  more  common  than  the  cor- 

n  prefixes  :    deci,  i-ioth  ;   cenli,   i-iooth;  Milli, 
ia,  10  times  ;  hecto,  100  times  ;  kilo,  1000  times  ; 


ENGLISH  MEASURES. 

ers,  1,000,000  microns,  39  3704  inches, 
nillimeter,  toW*0  meter,  ^-th  inch,  ap- 


proximately. 

Micron  ( ju 
(0.000039  inch 

Inch  (in. 


(Unit  of  Measure  in  Micrometry)  =  To'oo^n  mm.  Tooioooth  meter. 

2'oooth  inch,  approximately. 

25.399772  mm.  (25  4  mm.,  approx. ) 
Liter  —  1000  milliliters  or  icoo  cubic  centimeters,  i  quart  (approx.) 
Cubic  centimeter  (cc.  or  cctm. )  =  y^nrth  of  a  liter. 
Fluid  ounce  (8  Flui drachms)  =  29  578  cc.  (30  cc,  approx. ) 
Gram  =15  432  grains. 
Kilogram  (kilo)  =  2.204  avoirdupois  pounds  (2ith  pounds,  approx. ) 


Ounce  Avoirdupois  =  (4373   grains)  =  28.349  grams. 
•Ounce  Troy  or  Apothecaries  =  (480 grains)  =31. 103 grams. 


\ 


30  grams,  approx.) 


TEMPERATURE. 


To  change  Centigrade  to  Farenheit  :  (C.  Xf)+32  =  F.  For  example,  to  find 
the  equivalent  of  lo°  Centigrade,  C.  =  io°  (io°  X  f)  4  32  =  500  F. 

To  change  Farenheit  to  Centigrade  :  (  F.  —  320)  X|  =  C.  For  example,  to  re- 
duce 500  Farenheit  to  Centigrade,  F.  =50°,  and  (500  —  320)  X  f  =  io°  C.  ;  or  —  40 
Farenheit  to  Centigrade,  F.  =  —  40°  ( —  400  —  320)  =  —  720,  whence  —  720  X  § 
=  —  400  C. 

Address  of  Americau'Opticians  :  For  the  price  of  Microscopes  and  Microscopical  supplies,  the 
student  is  advised  to  obtain  a  catalog  of  one  or  more  of  the  opticians  named  below  For  the  cat- 
log  of  foreign  opticians,  see  addresses  in  the  table  of  tube-length,  p.  15.  Nearly  all  import  for- 
eign goods. 

The  Bausch  &  Lomb  Optical  Co., New  York  City  and  Rochester,  N.  Y. 

Rimer  and  Amend 205-211  Third  Avenue,  New  York  City 

The  Franklin  Educational  Co.,  ,  .       Harcourt  St.,  Boston,  Mass. 

].  Grunow; 70  West  39th  St.,  New  York 

The  Gundlach  Optical  Co., Rochester,  N.  Y. 

Wm.  Krafft,  (representative  of  Leitzin  America), 4 1 1  West  59th  St.,  New  York 

The  Mcintosh  Battery  and  Optical  Co 521-531  Wabash  Ave.,  Chicago,  111. 

Queen  &  Co..  Incorporated, 1010  Chestnut  St.,  Philadelphia,  Pa. 

Richards  &  Co.,  Limited, 30  E.  iSth  St.,  New  York,  10S  Lake  St,,  Chicago,  111. 

Edward  Pennock 3609  Woodland  Ave.,  Philadelphia,  Pa. 

Spencer  Lens  Co., 546  Main  St.,  Buffalo,  N.  Y. 

Walmsley,  Fuller  &  Co., 134-136  Wabash  Ave.,  Chicago,  111. 

Williams,  Brown  &  Earle, 10th  &  Chestnut  Sts.,  Philadelphia.  Pa. 

G.  S.  Woolman,  (Queen  &  Co.,  in  New  York), 116  Fulton  St.,  New  York 

J.  Zentmayer, . 209  South  nth  St.,  Philadelphia,  Pa, 


S00 139888 


Date  Due 

,' 


. 


Dept.  of  Botaj  jture. 


THE  MICROSCOPE 

AN 

INTRODUCTION    TO    MICROSCOPIC 
METHODS    AND    TO    HISTOLOGY 

BY    SIMON    HENRY    GAGE 

PROFESSOR  OF  MICROSCOPY,  HISTOL- 
OGY AND  EMBRYOLOGY  IN  CORNELL 
UNIVERSITY,  AND  THE  NEW  YORK 
STATE  VETERINARY  COLLEGE. 


7th 


EDITION    '"''%.        t&>  yd? 
REVISED 


A    3 


COMSTOCK  PUBLISHING  COMPANY 

ITHACA,    NEW    YORK 

1 899 


Copyright,  1899, 

By  Simon  Henry  Gage. 

All  Rights  Reserved. 


Printed  by 

Andrus  &  Church, 

Ithaca,  N.  Y. 


PREFACE  TO  THE  SEVENTH  EDITION. 


IN  this  edition  pp.  17  is,  49-52,  103  [04,  165-168,  that  is,  the  matter  pertain- 
ing to  numerical  aperture,  refraction,  the  filar  micrometer,  imbedding 
and  sectioning  by  the  paraffin  met  hod,  have  been  rewritten.  Some  errors  arc- 
noted,  and  additional  or  more  advantageous  methods  suggested  for  some  of 
the  sections.  The  title  page  has  been  slightly  altered  in  wording  to  make  it  more 
fully  indicate  the  aim  of  the  hook. 

Ithaca,  Jan.   11,   1S99. 


Corrections. — Pp.  33,  39,  46,  for  Pleurasigma,  read  Pleurosigma.  P.  97,  second 
paragraph,  change  No.  4  to  No.  3.  P.  159,  top  line,  change  carbol-xylene  to 
castor-xylene. 

Additions. — P.  145,  \  227  :  If  the  "proportions  of  acid  and  dicbromate  are 
greater,  this  cleaning  mixture  will  be  more  efficient.  The  following  from  Dr.  G. 
C.  Caldwell's  laboratory  guide  is  excellent.  Potassium  dichromate  40  grams: 
water  150  c.c;  sulphuric  acid  230  c.c.  For  preparing  this  mixture,  an  iron  kettle 
lined  with  heavy  sheet  lead  has  proved  both  satisfactory  and  economical. 

Pp.  154,  177,  \\  245,  30S.  Formaldehyde  dissociator  of  the  strength  of  2  c.c.  of 
formalin,  etc.,  to  the  liter,  has  been  found  more  satisfactory  than  5  c.c.  to  the 
liter. 

Pp.  160,  166,  \\  261,  275.  As  pointed  out  in  1S91  1  Proceed.  Amer.  Micr.  Soc, 
Vol.  xiii,  p.  82)  it  is  of  great  advantage  to  albumenize  the  slides  on  which  collo- 
dion sections  are  to  be  mounted.  This  is  done  by  placing  the  cleaned  slides  in  a 
jar  of  egg  albumen  1  to  200  of  water.  This  should  be  filtered  before  use.  After 
half  an  hour  or  more  the  slides  are  removed,  stood  on  end  on  a  towel  or  1 'lotting 
paper  and  allowed  to  dry  ;  they  can  then  he  stored  in  glass  jars  and  are  ready  to 
use  at  any  time.  From  such  albumenized  slides  the  collodion  sections,  fastened 
as  directed  in  2  261,  will  very  rarely  become  detached  even  with  repeated  manipu- 
lation. Such  albumenized  slides  are  excellent  for  use  with  paraffin  sections.  If 
the  sections  are  to  be  extended  with  warm  water  1  \  274  1  this  is  a  much  preferable 
method  to  that  witli  Mayer's  albumen. 

P.  160,  \\  257-259.  When  objects  are  imbedded  in  a  box  for  collodion  sections 
they  must  ordinarily  he  fixed  to  sonic-  holder  before  sectioning.  After  the  collo- 
dion is  hardened  in  chloroform  and  clarified,  remove  the-  paper  box,  absorb  the 
castor-xvlene  on  the  surface,  trim  the  end  and  put  sonic-  fresh,  thick  collodion  on 
the  cork  or  other  holder.  Press  the  imbedded  tissue  firmly  against  the  holder. 
Within  two  minutes  it  will  he  firmly  cemented  and  one  may  proceed  at  once  to 
clamp  the  holder  in  the  microtome  and  commence  cutting. 


w*y 


¥> 
x 


ADDITIONS. 


/J~ 


P.  i6o,  \  260.     For  handling  collodion  sections  the  better  quality  of  white  tissue 
paper  sold  by  stationers  has  been  found  excellent.     It  is  worth  while  to  have  it  cut 

into  pieces  about  60  X  50  mm. 

Pp.  171,  179,  ;  :;  292,  296,311.  For 
a  laboratory  it  has  been  found  ad- 
vantageous and  economical  to  furnish 
the  students  with  gummed  labels  for 
their  preparations.  The  form  used  is 
shown  in  the  accompanying  figure. 
Labels  of  this  kind  can  be  bought  in 
five  thousand  lots  for  35  to  40  cents 
per  thousand. 


J,lv<tr  aj. 


lOfl 


^ 


DATE 


Get*/ 


m 


VL 


& 


Also  for  temporary  storage,  and  for 
sets  of  preparations  to  be  issued  to 
students,  inexpensive  slide  drawers  fit- 
ting the  lockers  have  been  prepared. 
The  accompanying  figures  indicate  the 
consturction.  These  in  sizes  which 
will  hold  50  slides  (30x43  cm.)  cost 
from  $12  to  $15  per  100. 


\J 


=3 


P-  175.  %  258.  Grades  of  Alcohol.  It  has  been  found  by  careful  tests  that  quite 
accurate  percentages  of  alcohol  may  be  obtained  by  mixing  water  and  alcohol  as 
follows  :  Pour  alcohol  into  a  graduate  until  the  volume  of  alcohol  corresponds  to 
the  desired  percentage.  Add  water  until  the  volume  in  cubic  centimeters  corres- 
ponds to  the  original  percentage  of  the  alcohol  used.  For  example,  to  get  67% 
from  95  %  alcohol,  pour  67  c.c.  of  95  V  alcohol  into  a  graduate,  and  add  sufficient 
water  to  bring  the  volume  up  to  95  c.c.  For  50 %  alcohol  from  75%,  put  50  c.c. 
of  75%  alcohol  in  a  graduate,  add  sufficient  water  to  make  the  volume  75  c.c. 
From  the  change  in  volume  it  does  not  answer  to  mix  given  volumes  of  water  and 
alcohol  in  these  cases.  In  the  first  case,  if  one  mixed  75  c.c.  of  95^5  alcohol  and 
20  c.c.  of  water  the  resulting  mixture  won  d  be  over  75',  ;  but  if  sufficient  water 
is  added  to  bring  the  volume  back  to  the  original  percentage  more  than  20  c.c.  of 
water  is  added,  that  is  enough  more  to  compensate  for  the  shrinkage,  and  the 
result  is  approximately  accurate. 

P-  J75)  \  299-  ln  case  preparations  are  to  be  kept  some  time  in  alum  water,  2% 
of  chloral  hydrate  should  be  added  to  prevent  mold. 

P.  216,  §  360.  For  lettering  diagrams,  the  so-called  "easy  sign  markers" 
have  proved  very  satisfactory. 


PREFACE  TO  THE  SIXTH  EDITION. 


THE  rapid  advance  in  microscopical  knowledge,  and  the  great  strides  in  the 
sciences  employing  the  microscope  as  an  indispensable  tool,  have  reacted 
upon  the  microscope  itself,  and  never  before  were  microscopes  so  excellent,  con- 
venient and  cheap.  Indeed,  the  financial  reason  for  not  possessing  a  microscope 
can  no  longer  be  urged  by  any  high  school  or  academy,  or  by  any  person  whose 
profession  demands  it. 

Naturally,  to  get  the  greatest  good  from  instruments,  tools,  or  machines  of  any 
kind,  the  one  who  uses  them  must  understand  the  principles  upon  which  their 
action  depends,  their  possibilities  and  limitations. 

That  the  student  may  acquire  a  just  comprehension  of  some  of  the  fundamental 
principles  of  the  microscope,  and  gain  a  working  acquaintance  with  it,  this  book 
has  been  prepared.  It  is  a  growth  of  the  laboratory,  and  has  been  modified  from 
time  to  time  to  keep  pace  with  optical  improvements  and  advancing  knowledge. 

This  edition  has  been  largely  rewritten.  Many  new  figures  and  about  ninety 
pages  of  new  matter  have  been  added,  and  it  is  hoped  that  the  student  will  find  it 
a  real  help  in  his  efforts  to  become  master  of  the  modern  microscope. 


October  j/,  1S96. 


SIMON  HENRY  GAGE, 

Cornell  University. 


PREFACE  TO  THE  FIFTH  EDITION. 


THIS  edition  has  been  enlarged  nearly  one-half  by  the  elaboration  of  the  mat- 
ter in  the  previous  edition,  and  by  the  addition  of  a  wholly  new  chapter  on 
photo-micrography  and  on  photographing  natural  history  objects  in  a  hori- 
zontal  position  with  a  vertical  camera.  The  figures  have  been  distributed  in  the 
text,  and  many  new  ones  added. 

It  is  hoped  that  the  hook  as  it  now  appears  may,  while  remaining  strictly  ele- 
mentary, still  more  fully  meet  the  needs  of  those  who  wish  to  use  tbe  microscope 
for  serious  study  and  investigation.  The  aim  has  been  to  produce  a  book  for  be- 
ginners in  microscopy,  such  as  the  author  himself  felt  sorely  tbe  need  of  when  he 
began  tbe  study.  This  purpose  has  been  strengtbened  and  furthered  by  noting  the 
difficulties  of  the  various  classes  tbat  have  used  the  work  and  aided  in  its  evolution 
during  the  last  fifteen  years. 

The  author  wishes  to  acknowledge  the  aid  rendered  by  the  various  Optical  Com- 
panies for  information  freely  given,  and  for  the  loan  of  cuts  and  instruments 
(Bausch  &  Lomb  Optical  Co.,  Gundlach  Optical  Co.,  Queen  &  Co.,  and  all  the  op- 
ticians mentioned  in  the  table  of  tube-length,  p.  10).  I  feel  under  special  obliga- 
tion to  my  various  classes  for  the  enthusiasm  and  earnestness  with  which  they  have 
followed  the  instructions  in  the  book,  to  my  colleagues,  Professor  Wilder  and  In- 
structors Hopkins  and  Fish  for  suggestions,  to  Mrs.  Gage  for  criticising  the  manu- 
script, reading  proof,  preparing  tbe  index  and  the  original  figures,  to  Dr.  A.  C. 
Mercer  for  aid  in  preparing  tbe  chapter  on  photo-micrography,  to  Dr.  M.  D.  Ewell 
for  information  and  for  the  loan  of  apparatus,  and  finally,  to  many  other  friends 
who  have  used  the  previous  editions,  and  have  made  suggestions  whereby  it  is 
hoped  the  present  edition  is  greatly  improved. 

I  would  like  to  repeat  a  part  of  tbe  preface  to  the  tbird  and  to  the  fourth  editions, 
and  to  call  especial  attention  to  tbe  address  of  the  Hon.  J.  D.  Cox  at  the  recent 
meeting  of  tbe  American  Microscopical  Society:  "A  plea  for  systematic  instruc- 
tion in  the  technique  of  the  microscope  at  the  university,"  in  the  Proceedings  for 

Extract  from  the  preface  of  the  fourth  edition  : 

"The  author  would  feel  grateful  to  any  person  who  uses  this  book  if  he  would 
point  out  any  errors  of  statement  that  may  be  discovered,  and  also  suggest  modifi- 
cations whicb  would  tend  to  increase  the  intelligibility,  especially  to  beginners." 

From  the  tbird  edition  : 

"  It  is  thoroughly  believed  by  the  writer  that  simply  reading  a  work  on  the  mi- 
croscope, and  looking  a  few  times  into  an  instrument  completely  adjusted  by  an- 
other, is  of  very  little  value  in  giving  real  knowledge.  In  order  that  the  knowl- 
edge shall  be  made  alive,  it  must  be  made  a  part  of  the  student's  experience  by 
actual  experiments  carried  out  by  the  student  himself.  Consequently,  exercises 
illustrating  the  principles  of  the  microscope  and  the  methods  of  its  employment 
have  been  made  an  integral  part  of  the  work. 

"In  considering  the  real  greatness  of  the  microscope,  and  the  truly  splendid 


vi  PREFACE. 

service  it  has  rendered,  the  fact  has  not  been  lost  sight  of  that  the  microscope  is, 
after  all,  only  an  aid  to  the  eye  of  the  observer,  only  a  means  of  getting  a  larger 
image  on  the  retina  than  would  be  possible  without  it ;  but  the  appreciation  of  this 
retinal  image,  whether  it  is  made  with  or  without  the  aid  of  a  micro-scope,  must 
always  depend  upon  the  character  and  training  of  the  seeing  and  appreciating  brain 
behind  the  eye.  The  microscope  simply  aids  the  eye  in  furnishing  raw  material, 
so  to  speak,  for  the  brain  to  work  upon. 

"The  necessity  for  doing  a  vast  deal  of  drudgery,  or  'dead  work,'  as  it  has  been 
happily  styled  by  Professor  Leslie,  before  one  has  the  training  necessary  for  the 
appreciation  and  the  production  of  original  results,  has  been  well  stated  by  Beale  : 

"  'The  number  of  original  observers  emanating  from  our  schools  will  vary  as 
practical  work  is  favored  or  discouraged.  It  is  certain  that  they  who  are  most 
fully  conversant  with  elementary  details  and  most  clever  at  demonstration,  will  be 
most  successful  in  the  consideration  of  the  higher  and  more  abstruse  problems, 
and  will  feel  a  real  love  for  their  work  which  no  mere  superficial  inquirer  will  ex- 
perience. It  is  only  by  being  thoroughly  grounded  in  first  principles,  and  well 
practiced  in  mechanical  operations,  that  any  one  can  hope  to  achieve  real  success 
in  the  higher  branches  of  scientific  enquiry,  or  to  detect  the  fallacy  of  certain  so- 
called  experiments.'  n 

SIMON  HENRY  GAGE, 
Cornell  University, 

February  12,  189?.  Ithaca,  New  York,  U.  S.  A. 


CONTENTS 


CHAPTER  I. 

PA' 

\     I-  55 — The  Microscope  and  its  Parts — Demonstration  of  the  Function 

of  each  Part.     Figures  of  Laboratory  Microscopes, 1-32 

CHAPTER  II. 

\  56-119 — Lighting  and  Focusing,  Manipulation  of  Dry,  Adjustable,  and 
Immersion  Objectives;  Care  of  the  Microscope  and  of  the 
Eyes, 33-79 

CHAPTER  III. 
§120-144 — Interpretation  of  the  Appearances  under  the  Microscope,  .    .    .      80-91 

CHAPTER  IV. 
I  145-167— Magnification  of  the  Microscope  ;  Micrometry, 92-108 

CHAPTER  V. 
\  16S-178— Drawing  with  the  Microscope, 109-119 

CHAPTER  VI. 

\  179-218— rMicro-spectroscope  and  Micro-polariscope ;  Use  and  Applica- 
tion     120-139 

CHAPTER  VII. 

\  219-322— Slides  and  Cover-glasses  ;  Mounting  ;  Isolation  ;  Sectioning  by 
the  Collodion  and  Paraffin  Methods ;  Labeling  and  Storing 
Microscopical  Preparations  ;  Preparation  of  Reagents  ;  Ex- 
periments in  Micro-chemistry, .'    .     140-182 

CHAPTER  VIII. 

§322-351 — Photo-micrography  and  the  Photography  of  Natural  History 

Specimens  in  a  Horizontal  Position  with  a  Vertical  Camera,  .     183-209 

APPENDIX. 

I  352-370— The  use  of  Abbe's  Test-Plate  and  Apertometer 210 

Testing  Homogeneous  Liquids  ;  Experimental  Determination 
of  the  Equivalent  Focus  of  Objectives  and  Oculars  ;  Prepara- 
tion of  Diagrams  ;  Preparation  of  Drawings  for  Photo-engrav- 
ing,        213-219 

BOOKS  AND  PERIODICALS 210-225 

INDEX --: 


LIST  OF  ILLUSTRATIONS 


The  author  extends  grateful  acknowledgments  to  the  opticians  and  others  who 
have  loaned  cuts  for  this  edition.  The  source  of  each  figure  is  given  when  bor- 
rowed. The  other  figures  were  drawn  expressly  for  this  work  by  Mrs.  Cage.  Tin- 
frontispiece  was  drawn  by  Mr.  Gutsell,  of  the  University  Art  Department. 

J-'IG-  i'AGE. 

Frontispiece 

1-9.  The  principal  axis  and  center  of  various  lenses 2 

io-ri.  Principal  focus  with  converging  and  diverging  lenses 3 

12.  Chromatic  aberration 4 

13.  Spherical  aberration 4 

14-15.   Real  and  virtual  image  with  convex  lense3 5 

16.  Simple  microscope  and  eye  of  observer 6 

17.  Tripod  magnifier 7 

18.  Achromatic  triplet    The  Bausch  &  Lomb  Opt.  Co.)  .        .    .        7 

19.  Lens-holder  (The  Bausch  &  Lomb  Opt.  Co. ) 8 

20.  Dissecting  microscope  (The  Bausch  &  Lomb  Opt.  Co.) 9 

21.  Principle  of  the  compound  microscope 10 

22.  Dry  objective 11 

23.  Immersion  objective 12 

24.  Tube-length 15 

25.  Tube-length  when  nose  piece  and  Ocular  micrometer  are  used  (Zeiss'  cat- 

alog, No.  30) 16 

26.  Angular  aperture 17 

27-29.  Dry  and  immersion  objectives  (Ellenberger) 18 

30.  Section  of  Huygeniau  ocular  for  eye-point 22 

31.  Compensation  oculars  (Zeiss'  catalog,  No.  30) 24 

32.  Projection  oculars  (Zeiss'  catalog,  No.  30) 25 

33-34.  Ocular  micrometer  with  movable  scale  (Bausch  &  Lomb  Opt.  Co.)  .    .  25 

35.  Ocular  screw  micrometer  (Zeiss' catalog,  No.  30) 26 

36.  Triple  nose-piece  or  revolver  (Queen  &  Co. ) 27 

37.  Size  of  field  with  various  objectives  and  oculars 29 

38.  Principle  of  the  simple  microscope  (Fig.  16  repeated) 31 

39-40.  Dry  and  immersion  objectives  (Figs.  22-23  repeated) 34 

41.  Achromatic  condenser  (Zeiss' catalog,  No.  30) 41 

42-43.  Image  of  diaphragm  in  centering 42 

44-45.  Centering  the  source  of  illumination  on  the  object 43 

46-47,  Aperture  of  condenser  (from  Nelson) 13 

48-51.  Abbe  condenser,  central,  oblique  and  dark  ground  illumination  ....  47 

52.  Lamp  and  bull's  e3'e  condenser 49 

53~55-  Refraction  diagrams  (from  Carpenter-Dallinger) 50 

56.  Aberration  produced  by  the  cover-glass  (Ross) 58 


x  L  IS T  OF  ILL  US TRA  TIONS. 

57.  Cover  correction  by  changing  tube-length 54 

58.  Screen  for  face  and  microscope 56 

59.  Ward's  eye  shade  (Cut  loaned  by  Queen  &  Co.) 59 

60.  Double  eye-shade 59 

61-63.   Marker,  sectional  view  (Proc.  Amer.  Micr.  Soc,  1894) 64 

64-66.   Specimens  showing  the  use  of  the  marker 64 

67.  Krauss'  method  of  marking  objectives  on  a  nose-piece  (from  Dr.   Krauss, 

see  Proc.  Amer.  Micr.  Soc,  1895) 65 

68.  Removable  mechanical  stage  (Leitz  catalog)  .    .    "    ■ 65 

69.  Removable  mechanical  stage  (Bausch  &  Lomb  Opt.  Co.) 65 

70.  Zeiss'  Microscope  la  with  mechanical  stage  (Zeiss'  catalog,  No.  30)   ...  66 

71.  Watson  &  Sons,  Edinburgh,  student's  microscope  (Watson  &  Sons  catalog)  67 

72.  Nachet  et  Fils  microscope  No.  4  with  movable  stage  (cut  loaned  by  the 

Franklin  Educational  Co.) 68 

73.  BB  Microscope  of  the  Bausch  &  Lomb  Optical  Co.  (B  &  L) 69 

74.  Reichert's  microscope  Illb  (cut  loaned  by  Richards  &  Co. ) 70 

75.  Queen  &  Co.'s  microscope  II  of  the  continental  pattern  (Q.  &  Co.)  ...  71 

76.  Leitz'  microscope  lb  (cut  loaned  by  Wm.  Krafft) 72 

77.  Ross  eclipse  microscope  (cut  from  Walmsley,  Fuller  &  Co.) 73 

78.  AA.  Microscope  of  the  Bausch  &  Lomb  Optical  Co.  (B.  &  L.) 74 

79.  Beck's  star  microscope  (cut  loaned  by  Williams,  Brown  &  Earle)    ....  75 

80.  Zentmayer's  clinical  microscope  (Zentmayer) 76 

81.  Zentmayer's  microscope,  No.  V  (Zentmayer) 76 

82.  Leitz'  demonstration  microscope  (from  Win.  Krafft,  N.  Y. ) 77 

83.  Leitz'  microscope  IV  (from  Wm.  Krafft,  N.  Y.) 77 

84.  Queen  &  Co.'s  acme  microscope,  No.  IV  (Q.  &  Co.) 78 

S5.  Mcintosh's  scientific  microscope,  No.  2  (Mcintosh  Battery  Co.) 79 

86.  Letters  mounted  in  stairs  to  show  order  of  coming  into  focus 82 

87.  Putting  on  a  cover-glass 84 

88.  Oil  and  air  bubbles 85 

89.  Glass  rods  in  optical  section 86 

90.  Double  contour 87 

91.  Micrometer  with  ring  to  facilitate  finding  the  lines 94 

92.  Wollaston's  camera  lucida 95 

93.  Geometrical  diagram  showing  size  of  object  and  image 96 

94.  Image  and  object  with  differing  tube-length 96 

95.  Standard  distance  for  magnification  with  Wollaston's  camera  lucida  ...  98 

96.  Standard  distance  for  magnification  with  the  Abbe  camera  lucida    ....  98 

97.  Preparation  of  blood  corpuscles  with  ring  around  a  group 101 

98-99.  Ocular  micrometer  (Figs  33-34  repeated) 103 

100.  Ocular  screw  micrometer  (Fig.  35  repeated) 104 

101.  Lines  of  stage  and  ocular  micrometer  iu  getting  the  valuation  of  the  ocu- 

lar micrometer 107 

102.  Abbe  camera  lucida  with  450  mirror no 

103.  Geometrical  figure  going  with  Fig.  102 no 

104.  Ocular  showing  eye-point  (Fig.  30  repeated) no 

105.  Wollaston's  camera  lucida  (Fig.  92  repeated) in 

J06.  Abbe  camera  lucida  with  350  mirror 114 

107.  Geometrical  figure  going  with  Fig.  106 114 

.   Upper  view  of  the  prism  of  the  Abbe  camera  lucida 114 


L  1ST  OF  ILL  TS TRA  TIONS.  x i 

09.  Quadrant  attached  to  the  mirror  of  the  Abbe  camera  lucida 114 

10.  Inclined  microscope  with  the  Abbe  camera  lucida 115 

ri.    Drawing  hoard  fur  the  Abbe  camera  (The  Bausch  &  I,omb  Opt.  Co.      .  116 

12.  Micrometer  lines  indicating  the  scale  of  a  drawing us 

13.  Longisection  of  the  Abbe  micro-spectroscope  (cut  loaned  by  the  Bausch 

&  I.omb  Opt.  Co.) 121 

14.  Slit  mechanism  of  the  micro-spectroscope  (from  B.  &L.) 121 

[5.   Various  spectrums 122 

16.  Absorption  spectrum  of  hemoglobin,  etc.  (Gamgee  &  MeMunn)   ....  124 

17.  Section  of  the  micro-spectroscope 126 

18.  Prism  showing  apparent  reversal  of  colors 126 

19.  Section  of  a  micro-polariscope 126 

20.  Micrometer  calipers  (Brown  .S:  Sharp) [43 

21.  Cover-glass  measurer  (The  Bausch  &.  Lomb  Opt.  Co.) 144 

22.  Zeiss  cover-glass  measurer  (from  Zeiss' catalog) 14c 

23.  Putting  on  a  cover-glass  |  Fig.  S7  repealed) 146 

24.  Needle  holder  (Queen  &  Co.) 146 

25.  Turn  table  (Queen  &  Co.) 14S 

26.  Centering  card 140. 

27.  Anchoring  a  cover-glass    .....             j^o 

28.  Irrigation,  staining,  etc.,  under  the  cover 150 

29.  Moist  chamber  for  fibrin,  blood  corpuscles,  eic.   (from  Proc.  Amer.  Micr. 

Soc,  1891) 151 

30.  Adjustable  lens  holder  (Leilz,  cut  from  Win.  Krafft) 155 

31.  Adjustable  lens-holder  (The  Bausch  &  I.omb  Opt,  Co.) 156 

32.  Preparation  vials  (Proc.  Amer.  Micr.  Soc,  1S95) 159 

33.  Pipette  for  stains,  etc.  (Whitall,  Tatum  &  Co.) 161 

34.  Waste  bowl  with  rack  and  funnel  (cut  loaned  by  Wm.  Wood  6c  Co.  1    .    .  162 

35.  Round  aquarium  for  waste  bowl,  rinsing  jar,  etc.  (Whitall,  Tatum  &  Co.  |  162 

36.  Glass  box  for  cleaning  slides  and  covers  (Whitall,  Tatum  6c  Co.)  .     .    .    .  162 

37.  Balaam  bottle 164 

38.  vSerial  section  slide,  showing  order  of  arranging  sections 170 

39.  Writing  diamond  (Queen  &  Co. ) 173 

40.  Drawer  of  cabinet  for  slides  (Proc.  Amer.  Micr.  Soc,  1883) 174 

41.  Cabinet  for  microscopical  specimens  |  Proc.  Amer.  Micr.  Soc,  1883).    .    .  174 

42.  Czapski's  iris  diaphragm  ocular  (  Zeiss'  catalog,  No.  30) 1S1 

43.  Walmsley's  large  photo- mierographic  camera  (from  Mr.  Walmsley)  .    .    .  1S6 

44.  I„eitz'  vertical  photo  mierographic  camera  (from  Wm.  Krafft) [88 

45.  Projection  oculars  (Fig.  32  repeated) [89 

46.  Walmsleys  autograph  camera  in  a  vertical  position  (from  Mr.  Walmsley)  190 

47.  Same  in  horizontal  position 192 

48.  Vertical  photo-micrographic  camera  (the  Bausch  <S:  I.omb  Opt.  Co.)   .  193 

49.  Zeiss'  70  millimeter  projection  objective  (from  Zeiss'  photo-micrographic 

catalog) 195 

50.  Focusing  screen 195 

51.  Perigraphic  photographic  objective  (The  Gundlach  Opt  Co.) 196 

52.  Zeiss  anastigmatic  photographic  objective  (from  the  Bausch  and   I.omb 

Opt.  Co.  ) 196 

53.  Focusing  glass  (from  the  Gundlach  Opt.  Co.) \u~ 

54.  The  tripod  as  a  focusing  glass 198 


x ii  L  1ST  OF  ILL  USTRA  TIONS. 

155.  Engraving  glass  (The  Bausch  &  Lomb  Opt.  Co.) 19S 

156.  Hausch  &  Lonib's  chain  lens-holder  for  use  with  a  dissecting  lens,  hold- 

ing an  engraving  glass,  etc.  (B.  &  L. ) 199 

157.  Bull's  eye  and  lamp  (Fig.  52  repeated) 2co 

[58.   Zeiss'  vertical  photo-micrographic  camera  (Zeiss'  catalog) 202 

159.   Rack  for  drying  negatives  (Rochester  Opt.  Co. ) 203 

160-161.  Sections  of  the  head  and  brain  of  Diemyctylus  (Mrs.  Gage,  from  the 

Wilder  Quarter  Century  Book) 203 

162.  Vertical  photographic  camera  for  picturing  brains  and  other  preparations 

in  a  horizontal  position 207 

164.  Abbe's  test  plate 211 

165.  Abbe's  apertotneter  (Zeiss' catalog^ 212 


THE    MICROSCOPE    IN    SECTION. 


i.  Compensation  ocular   ■    12;  it   is  a  positive 

ocular. 
2.  Draw-tube,  by  which  the  tube  is  lengthened 

or  shortened. 

5.  .Main  tube  or  body,  to  the  lower  end  of  which 

the  objective  or   revolving  nose-piece  is 
attached. 
i    Society   screw   in   the   lower    end   of    the 

draw-tube. 
Society    screw    in    the    lower    end    of    the 
tube. 

6.  <  ibjective  in  position. 

7    Stage,  under  which  is  the  substage  w  ith  the 
substage  condenser. 
Spring  clip  for  holding  the  specimen 


9.  Screw  for  centering,  and  handle  of  the 
iris  diaphragm  in  the  achromatic  con- 
denser (see  Fig.  41). 

10.  Iris  diaphragm  outside  the  principal  focus 

of   the   condenser   for   use   in   centering 

{$  77) 

11.  Mirror  with  plane  and  concave  faces. 
12    Horse-shoe  base. 

13.  Rack  and  pinion  for  the  substage  conden- 
ser. 

1  ).   Flexible  pillar. 

15.  Part  of  pillar  with  spiral  spring  of  fine 
adjustment. 

16    Screw  of  fine  adjustment. 

i;    Milled  bead  of  coarse  adjustment. 


THE    MICROSCOPE 


A  X  I ) 


MICROSCOPICAL    METHODS 


CHAPTER  I. 


THE  MICROSCOPE  AND  ITS  PARTS. 


APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER. 

A  simple  microscope  (?  2,  9)  ;  A  compound  microscope  with  nose-piece  (Figs. 
6S-80),  eye-shade  (Figs.  59-60),  achromatic  (g  18),  apochromatic  {\  20),  dry  {\  15), 
immersion  {\  16),  unadjustable  and  adjustable  objectives  (§  21,  22),  Huygenian  or 
negative  {\  35),  positive  (y\  34)  and  compensation  oculars  (?  36),  stage  microme- 
ter, homogeneous  immersion  liquid  ($  16,  Ch.  IV),  benzin  and  distilled  water  {\  103- 
10S).     Mounted  letters  or  figures  [\  49)  ;  ground-glass  and  lens  paper  (§  49). 

A  MICROSCOPE. 

§  I.  A  Microscope  is  an  optical  apparatus  with  which  one  may  obtain  a  clear 
image  of  a  near  object,  the  image  being  always  larger  than  the  object ;  that  is,  it 
enables  the  eye  to  see  an  object  under  a  greatly  increased  visual  angle,  as  if  the  ob- 
ject were  brought  very  close  to  the  eye  without  affecting  the  distinctness  of  vision. 
Whenever  the  microscope  is  used  for  observation,  the  eye  of  the  observer  forms  an 
integral  part  of  the  optical  combination  (Figs.  16,  21). 

\  2.  A  Simple  Microscope. — With  this  an  enlarged,  erect  image  of  an  object 
may  be  seen.  It  always  consists  of  one  or  more  converging  lenses  or  lens-systems 
(Figs.  16-20),  and  the  object  must  be  placed  within  the  principal  focus  { \  9).  The 
simple  microscope  may  be  held  in  the  hand  or  it  may  be  mounted  in  some  way  to 
facilitate  its  use  (Figs.  17-20). 

N.  C.  State  College 


MICROSCOPE  AND  ACCESSORIES. 


{CH.  I. 


d  cd% 


Figs.  1-9,  showing  the  Principal  Optic  Axis  and  the  Optical  Center  of  various 
forms  of  Lenses. 

Axis.   The  Principal  Optic  Axis,     c-c' '.  Centers  of  curvature  of  the  tzvo  surfaces 


of  the  lens.     c.  I.   Optical  center  of  the  lens, 
lens  surfaces,     t-t' .   Tangents  in  Fig.  4. 


r-r' '.  Radii  of  curvature  of  the  two 


\  3.  Principal  Optic  Axis. — In  spherical  lenses,  i.  e.,  lenses  whose  surfaces  are 
spherical,  the  Axis  is  the  part  of  the  line  joining  the  centers  of  curvature  and 
traversing  the  lens  ;  it  is  the  unbroken  part  of  the  line  c-c'  in  all  the  figures.  In 
lenses  with  one  plane  surface  (Figs.  3,  6,  7)  the  radius  of  the  plane  surface  is  any  line 
at  right  angles  to  it,  but  in  determining  the  axis  it  must  be  the  one  which  is  con- 
tinuous with  the  radius  of  the  curved  surface,  consequently  the  axis  in  such  lenses 
is  on  the  radius  of  the  curved  surface  which  meets  the  plane  surface  at  right  angles. 

§  4.  Optical  Center. — The  optical  center  of  a  lens  is  the  point  through  which  rays 
pass  without  angular  deviation,  that  is,  the  emergent  ray  is  parallel  to  the  incident 
ray.  It  is  determined  geometrically  by  drawing  parallel  radii  of  the  curved  sur- 
faces, r-rr  iu  Figs.  4-9,  and  joining  the  peripheral  ends  of  the  radii.  The  optical 
center  is  the  poiut  on  the  axis  cut  by  the  line  joining  the  radii.     Iu  Figs.  4-5  it  is 


CH.  /.] 


J//<  V/(  >S<  'OPE  AND  .h  '<  ESSi  'AV/.'.V. 


within   the  lens  ;  in  6-7  it  is  at  the  curved  surface,  and  in  the  meniscus  it  is 

wholly  outside  the  U-ns,  being  situated  on  the  side  <>t  the  greater  curvature. 

In  determining  the  center  in  a  lens  with  a  plane  surface,  the  conditions  can  he 
satisfied  only  by  using   the   radius  of  the  curved  surface  which  is  continuous  with 
the  axis  of  the  lens,  then  any  line  at  right  angles  to  the  plane  surface  will  he  ; 
allel  with  it,  and  may  he  considered  part  of  the  radius  of  the  plane  surface.     (That 
is,  a  plane  surface  may  he  considered   part  of  re   with   infinite   radius,  hence 

any  line  meeting  the  plane  surface  at  right  angles  may  he  considered  as  the 
peripheral  part  of  the  radius.)  In  Figs.  6,  7,  ( r')  is  the  radius  of  the  curved  sur- 
face and  (r)  of  the  plane  surface  ;  and  the  point  where  a  line  joining  the  ends  of 
these  radii  crosses  the  axis  is  at  the  curved  surface  in  each  case. 

By  a  study  of  Fig.  4  it  will  he  seen  that  if  tangents  he  drawn  at  the  peripheral 
ends  of  the  parallel  radii,  the  tangents  will  also  be  parallel  and  a  ray  incident  at  one 
tangential  point  and  traversing  the  lens  and  emerging  at  the  other  tangential  point 
acts  as  if  traversing,  and  is  practically  traversing  a  piece  of  glass  which  has  paral- 
lel sides  at  the  point  of  incidence  and  emergence,  therefore  the  emergent  ray  will 
be  parallel  with  the  incident  ray.  This  is  true  of  all  rays  traversing  the  center  of 
the  lens. 

\  5.  Secondary  Axis.— Every  ray  traversing  the  center  of  the  lens,  except  the 
principal  axis,  is  a  secondary  axis  ;  and  every  secondary  axis  is  more  or  less  oblique 
to  the  principal  axis.  In  Fig.  14,  line  (  2),  is  a  secondary  axis,  and  in  Fig.  15,  line 
(1).     See  also  Fig.  57. 


Figs.  10,  11. — Sectional  views  of  a  con- 
cave or  diverging  and  a  convex  or  con-'' 
verging  lens  to  s/iozo  that  in  the  concave 
lens  the  principal  focus  is  virtual  as  indi- 
cated by  the  dotted  lines,  -while  with  the 
convex  lens  the  focus  is  real  and  on  the 
side  of  the  lens  opposite  to  that  front  which 
the  light  comes.  The  principal  focal  dis- 
tance is  the  distance  along  the  axis,  from 
the  optical  cetiter  to  the  principal  focus 


^ 

\    * 

j,   w 

■ 

/ 

i 

\ 

\ 

i 

1 
$ 

F 

/ 

II 


\  6.  Principal  Focus.  —  This  is  the  point  where  parallel  rays  traversing  the  lens 
cross  the  axis  ;  and  the  distance  from  the  focus  to  the  center  of  the  lens  measured 
along  the  axis  is  the  Principal  Focal  Distance.  In  the  diagrams,  Fig.  ro  is  seen 
to  he  a  diverging  lens  and  the  rays  cross  the  axis  only  by  being  projected  hack- 
ward.  Such  a  focus  is  said  to  he  virtual,  as  it  it  has  no  rial  existence.  In  Fig.  11 
the  rays  do  cross  the  axis  and  the  focus  is  said  to  he  real.  If  the  light  came  from 
the  opposite  direction  it  would  he  seen  that  there  is  a  principal  focus  on  the  other 
side,  that  is  there  are  two  principal  foci,  one  on  each  side  of  the  lens.  These  two 
foci  are  both  principal  foci  ;  and  as  there  may  be  foci  on  secondary 
each  focus  on  a  secondary  axis  has  its  conjugate.  In  the  formation  ol  images  the 
image  is  the  conjugate  of  the  object  and  conversely  the  object  is  the  conjugate  of 
the  image. 


MICROSCOPE  AND  ACCESSORIES. 


\CH.  I. 


Fig.  12. — Double  Convex  Lens,  Showing  Chromatic  Aberration. 

The  ray  of  white  light  (w)  is  represented  as  dividing  into  the  short  waved,  blue 
(b)  and  the  long  waved,  red  (r)  light.  The  blue  (b)  ray  comes  to  a  focus  nearer 
the  lens  and  the  red  ray  (r)  farther  from  the  lens  than  the  principal  focus  (/). 
Principal  focus  (f )  for  rays  very  near  the  axis,  f  andf,  foci  of  blue  and  red 
light  coming  from  near  the  edge  of  the  lens.  The  intermediate  wave  lengths 
would  have  foci  all  the  way  between  f  andf". 


§  7.  Chromatic  Aberration. — This  is  due  to  the  fact  that  ordinary  light  consists 
of  waves  of  varying  length,  and  as  the  effect  of  a  lens  is  to  change  the  direction  of 
the  waves,  it  changes  the  direction  of  the  short  waves  more  markedly  than  the 
long  waves.  Therefore  the  short  waved,  blue  light  will  cross  the  axis  sooner  than 
the  long  waved,  red  light,  and  there  will  result  a  superposition  of  colored  images, 
none  of  which  are  perfectly  distinct.     (Fig.  12). 


Fig.  13.  The  ray  (o)  near  the 
edge  of  the  lens  is  brought  to  a 
focus  nearer  the  lens  than  the 
ray  (i).  Both  are  brought  to 
a  focus  sooner  than  rays  very 
near  the  axis,  (f )  Princi- 
pal focus  for  rays  very  near 
the  axis  ;  {f)  Focus  for  the 
ray  (i),  and  (f")  Foctis  for 
the  ray  (o).  Intermediate 
rays  would  cross  the  axis  all 
the  way  from  {f"  tof). 


Fig.  13. 


Double  Convex  Lens,  showing 
Spherical  Aberration. 


§  8.  Spherical  Aberration. — This  is  due  to  the  unequal  turning  of 
the  light  in  different  zones  of  a  lens.  The  edge  of  the  lens  refracts 
proportionally  too  much  and  hence  the  light  will  cross  the  axis  or  come 
to  a  focus  nearer  the  lens  than  a  ray  which  is  nearer  the  middle  of  the 
lens.  Thus,  in  Fig.  13,  if  the  focus  of  parallel  rays  very  near  the  axis 
is  at/,  rays  (0  i)  nearer  the  edge  would  come  to  a  focus  nearer  the 
lens,  the  focus  of  the  ray  nearest  the  edge  being  nearest  the  lens. 
Every  simple  lens  has  the  defect  of  both  chromatic  and  spherical  aber- 
ration, and  to  overcome  this,  kinds  of  glass  of  different  refractive  power 


CII.  /.] 


MICROSCOPE  AND  ACCESSORIES. 


and  different  dispersive  power  are  combined,  concave  lenses  neutraliz- 
ing the  defects  of  convex  lenses.  If  the  concave  lens  is  not  sufficiently 
strong  to  neutralize  the  aberration  of  the  convex  lens,  the  combination 
is  said  to  be  under-corrected,  while  if  it  is  too  strong  and  brings  the 
marginal  rays  or  the  blue  rays  to  a  focus  beyond  the  true  principal 
focus,  the  combination  is  over-corrected. 

Probably  no  higher  technical  skill  is  used  in  any  art  than  is  requisite 
in  the  preparation  of  microscopical  objectives,  oculars  and  illuminators. 


Figs.  14  and  15.  14.  Convex  lens 
showing  the  position  of  the  object  (A-B) 
outside  the  principal  focus  {E),  and 
the  course  of  the  rays  in  the  formation 
of  real  images.  To  avoid  confusion 
the  rays  are  drazvn  from  only  one  point. 

A  B.  Object  outside  the  principal  fo- 
cus. B'  A' ' .  Real,  enlarged  image  on 
the  opposite  side  of  the  lens. 

Axis.  Principal  optic  axis.  1,  2,  3. 
Rays  after  traversing  the  lens.  They 
are  converging,  and  consequently  form 
a  real  image.  The  dotted  line  and  the 
line  (2)  give  the  direction  of  the  rays 
as  if  unaffected  by  the  lens.  (E).  The 
pri>icipal  focus. 

Fig.  15.  Convex  lens  showing  the  po- 
sition of  the  object  {A  B)  within  the 
principal  focus  and  the  course  of  rays 
in  the  formation  of  a  virtu,  I  image. 


14. 


15- 


A  B.  The  object  placed  between  the  lens  and  its  focus ;  A/  B'  virtual  image 
formed  by  tracing  the  rays  backivard.  It  appears  on  the  same  side  of  the  lens  as 
the  object,  and  is  erect  (?  0). 

A  a  is.   The  principal  optic  axis  of  the  lens.     F.   The  principal  focus. 

/,  2,  3.  Rays  from  the  point  B  of  the  object.  They  are  diverging  after  travers- 
ing the  lens,  but  not  so  divergent  as  if  no  lens  were  present,  as  is  shown  by  the 
dotted  lines.  Ray  {/)  traverses  the  center  of  the  lens,  and  is  therefore  not  deviated. 
It  is  a  secondary  axis  (§5). 


\  8a.  Geometrical  Construction  of  Images. — As  shown  in  Figs.  14-15,  for  the 
determination  of  any  point  of  an  image,  or  the  image  being  known,  to  determine 
the  corresponding  part  of  the  object,  it  is  necessary  to  know  the  position  of  the 
principal  focus  (and  there  is  one  on  each  side  of  the  lens,  \  6),  and  the  opt: 
center  (Figs.  1-9)  of  the  lens.  Then  a  secondary  axis,  (2)  in  Fig.  14,  (l)  in  Fig. 
15,  is  drawn  from  the  extremity  of  the  object  and  prolonged  indefinitely  above  the 
lens,  or  below  it  for  virtual  images.  A  second  line  is  drawn  from  tin-  extremity  of 
the  object,  (3)  in  Fig.  14,  (2)  in  Fig.  15,  to  the  lens  parallel  with  the  principal 
axis.     After  traversing  the  lens  it  must  be  drawn  through  the  principal  focal  point. 


MICROSCOPE  AND  ACCESSORIES. 


\_CH.  I. 


If  now  it  is  prolonged  it  will  cross  the  secondary  axis  above  the  lens  for  a  real 
image  and  below  for  a  virtual  image.  The  crossing  point  of  these  lines  determines 
the  position  of  the  corresponding  part  of  the  image.  Commencing  with  any  point 
of  the  object  the  corresponding  point  of  the  image  ma}'  be  determined  as  just 
described,  and  conversely  commencing  with  the  image  corresponding  points  of 
the  object  may  be  determined. 

SIMPLE   MICROSCOPE  :    EXPERIMENTS. 

§  9.  Employ  a  tripod  or  other  simple  microscope,  and  for  object  a 
printed  page.  Hold  the  eye  about  two  centimeters  from  the  upper  sur- 
face of  the  magnifier,  then  alternately  raise  and  lower  the  magnifier 
until  a  clear  image  may  be  seen.  (This  mutual  arrangement  of  micro- 
scope and  object  so  that  a  clear  image  may  be  seen,  is  called  focusing). 
When  a  clear  image  is  seen,  note  that  the  letters  appear  as  with  the 
unaided  eye  except  that  they  are  larger,  and  the  letters  appear  erect 
or  right  side  up,  instead  of  being  inverted,  as  with  the  compound  mi- 
croscope (§  10,  49). 


Fig.  16.  Diagram  of  the  simple  microscope  show- 
ing the  course  of  the  rays  and  all  the  images,  and 
that  the  eye  forms  an  integral  part  of  it. 

A1  B\  The  object  within  the  principal  focus.  A3 
B'\  The  virtual  image  on  the  same  side  of  the  lens 
as  the  object.  It  is  indicated  with  dotted  lines,  as  it 
has  no  actual  existence. 

B1  A.  Retinal  image  of  the  object  {A1  B).  The 
vittual  image  is  simply  a  projection  of  the  retinal 
image  hi  the  field  of  vision. 

Axis.  The  principal  optic  axis  of  the  microscope 
and  of  the  eye.  Cr.  Cornea  of  the  eye.  L.  Crystal- 
line lens  of  the  eye.  R.  Ideal  refracting  surface  at 
which  all  the  refractions  of  the  eye  may  be  assumed 
to  take  place. 


Hold  the  simple  microscope  directly  toward  the  sun  and  move  it 
away  from  and  toward  a  piece  of  printed  paper  until  the  smallest 
bright  point  on  the  paper  is  obtained.  This  is  the  burning  point 
or  focus,  and  as  the  rays  of  the  sun  are  nearly  parallel,  the  burning 


CH.  /.] 


MICROSCOPIC  AND  ACCESSORIES. 


Fig.  17. 
Tripod  Ma gnij 


point  represents  approximately  the  principal  focus  (  Pigs.  10,  n).  With- 
out changing  the  position  of  the  paper  or  the  magnifier,  look  into  the 
magnifies  and  note  that  the  letters  arc  very  in- 
distinct or  invisible.  Move  the  magnifier  a  cen- 
timeter or  two  farther  from  the  paper  and  no 
image  can  be  seen.  Now  move  the  magnifier 
closer  to  the  paper,  that  is,  so  that  it  is  less  than 
the  focal  distance  from  the  paper,  and  the  letters 
will  appear  distinct.  This  shows  that  in  order 
to  see  a  distinct  image  with  a  simple  microscope, 
the  object  must  always  be  nearer  to  it  than  its 
principal  focal  point.  Or,  in  other  words,  the 
object  must  be  within  the  principal  focus.  Com- 
pare (§  49.). 

After  getting  as  clear  an  image  as  possible  with  a  simple  microscope, 
do  not  change  the  position  of  the  microscope  but  move  the  eye  nearer 
and  farther  from  it,  and  note  that  when  the  eye  is  in  one  position,  the 
largest  field  may  be  seen.  This  position  corresponds  to  the  eye-point 
(Fig.  30)  of  an  ocular,  and  is  the  point  at  which  the  largest  number  of 
rays  from  the  microscope  enter  the  eye.  Note  that  the  image  appears 
on  the  same  side  of  the  magnifier  as  the  object. 

Simple  microscopes  are 
very  convenient  when 
only  a  small  magnifica- 
tion (Ch.  IV)  is  desired, 
as  for  dissecting.  Achro- 
matic triplets  are  excel- 
lent  and  convenient  for 

Fig.  18.  Achromatic  Triplet  for  the  pocket.  As  show/!  in  the  left  hand  figure  it 
is  composed  of  three  lenses,  one  of  crown  and  l?co  of  flint  glass.  The  whole  is 
protected  by  a  metal  covering  when  not  in  use.     (Bait sch  &  Lomb  Opt.  Co.  1 

the  pocket  (Fig.  18).  For  use  in  conjunction  with  a  compound  micro- 
scope, the  tripod  magnifier  (Fig.  17)  is  one  of  the  best  forms.  For 
many  purposes  a  special  mechanical  mounting  like  that  of  Figs.  [9,  20, 
is  to  be  preferred. 

COMPOUND   MICROSCOPIC. 


\  io.   A  Compound  Microscope. — This  enables  one  to  see  an  enlarged,  inverted 
image.     It  always  consists  of  two  optical  parts — an  object  ii<  "luce  an   en- 

larged, inverted,  real  image  of  the  object,  and  an  ocular  acting  in  general  like  a 
simple  microscope  to  magnify   this  real   image  (Fig.  2l).     Then  >  usually 


8 


MICROSCOPE  AND  ACCESSORIES. 


[CH.  I. 


present  a  mirror,  or  both  a  mirror  and  some  form  of  condenser  or  illuminator  for 
lighting  the  object.  The  stand  of  the  microscope  consists  of  certain  mechanical 
arrangements  for  holding  the  optical  parts  and  for  the  more  satisfactory  use  of 
them.     (See  frontispiece). 


Fig.  19.  Lens  Holder  with  adjustments  for  focusing  and  for  turning  the  lens  in 
any  direction.  This  is  especially  useful  in  dissecting  the  minute  parts  of  animals 
too  large  for  the  regular  dissecting  microscope  {Fig.  20).     (Bausch  &  Lomb  Opt.  Co.). 


Fig.  20.   Dissecting  Microscope  roil/i  hand  rests  and  nose-piece  for  scleral  lens* 
different />ower.     (Bausc/i  &  Lomb  Optical  Co.). 


IO 


MICROSCOPE  AND  ACCESSORIES. 


[CH.  I. 


MECHANICAL   PARTS. 


\  ii.  The  Mechanical  Parts  of  a  laboratory,  compound  microscope  are  shown  in 
the  frontispiece,  and  are  described  in  the  explanation  of  that  figure.  The  student 
should  study  the  figure  with  a  microscope  before  him  and  become  thoroughly 
familiar  with  the  names  of  all  the  parts.  See  also  the  cuts  of  microscopes  at  the 
end  of  Ch.  II. 

OPTICAL   PARTS. 

\  12.  Microscopic  Objective.  —  This 
consists  of  a  converging  lens  or  of  one 
or  more  converging  lens-systems,  which 
give  an  enlarged,  inverted,  real  image  of 
the  object  (Figs.  14,  21).  And  as  for  the 
formation  of  real  images  in  all  cases, 
the  object  must  be  placed  outside  the 
principal  focus,  instead  of  within  it,  as 
for  the  simple  microscope.  (See  #§  9, 
49,  Figs.  16,  21). 

Modern  microscopic  objectives  usu- 
ally consist  of  two  or  more  s}  stems  or 
combinations  of  lenses,  the  one  next 
the  object  being  calkd  the  front  com- 
bination or  lens,  the  one  farthest  from 
the  object  and  nearest  the  ocular,  the 
back  combination  or  system.  There  may 
be  also  one  or  more  intermediate  sys- 
tems. Each  combination  is,  in  general, 
composed  of  a  convex  and  a  concave 
lens.  The  combined  action  of  the  sys- 
tems serves  to  produce  an  image  free 
from  color  and  from  spherical  distor- 
tion. In  the  ordinary  achromatic  ob- 
jectives the  convex  lenses  are  of  crown 
and  the  concave  lenses  of  flint  glass 
(Figs.  22,  23). 

Fig.  21.  Diagram  shozuing  the  prin- 
ciple of  a  compound  microscope  with 
the  course  of  the  rays  f  om  the  object 
(A  B)  through  the  objective  to  the  real 
image  (B/  A'),  thence  through  the  ocu- 
lar and  into  the  eye  to  the  retinal  image 
(A2  B2),  and  the  projection  of  the  retinal 
image  into  the  field  of  vision  as  the 
virtual  image  {B3  A3). 

AB.  The  object.  A2B2.  The  retinal 
im  age  of  the  inverted  real  image, (BlAx), 
formed  by  the  objective.  B3A3.  The  in- 
verted virtual  image,  a  projection  of 
the  retinal  image. 


CI  I.  /.] 


MicA'osa  )/'/■:  .ixi)  accessories. 


i  i 


A  vis.   The  principal  optic  axis  of  the  microscope  ami  of  the  - 

Cr.  Cornea  of  the  eye.  1..  Crystalline  lens  of  the  eye.  A'.  Single,  ideal,  re- 
fracting surf  ace  at  which  all  the  refractions  of  the  eye  may  be  assumed  to  take 
place. 

F.  /•'.    The  principal  focus  of  thepositive  ocular  and  of  the  obja  it 

Minor.  The  mirror  reflecting  parallel  rays  to  the  object.  'The  light  is  central. 
See  Ch.  If. 

Pos.  Ocular.  An  ocular  in  which  the  real  image  is  formed  outside  the  ocular. 
Compare  the  positive  ocular  '.villi  Tie  simple  mieroscope  {Fig.  16). 

NOMENCLATURE   OR   TERMINOLOGY   OF   OBJECTIVES. 

§  r3  Equivalent  Focus.  —  In  America,  England,  and  sometimes  also  on  the  Con- 
tinent, objectives  are  designated  by  their  equivalent  focal  length.  This  length  is 
given  either  in  inches  (usually  contracted  to  in.)  or  in  millimeters  (  mm  |.  Thus  : 
An  objective  designated  fa  in.  or  2  mm.,  indicates  that  the  objective  produces  a 
real  image  of  the  same  size  as  is  produced  by  a  simple  converging  lens  whose  prin- 
cipal focal  distance  is  ra5  inch  or  2  millimeters  (Fig.  n).  An  objective  marked  ; 
in.  or  75  mm.,  produces  approximately  the  same  sized  real  image  as  a  simple  con- 
verging lens  of  3  inches  or  75  millimeters  focal  length.  And  in  accordance  with 
the  law  that  the  relative  size  of  object  and  image  vary  directly  as  their  distance 
from  the  center  of  the  lens  (Figs  14.  15,  see  Ch.  IV,)  it  follows  that  the  less  the 
focal  distance  of  the  simple  lens  or  of  the  equivalent  focal  distance  of  the  objec- 
tive, the  greater  is  the  size  of  the  real  image,  as  the  tube-length  remains  constant 
and  the  image  in  all  cases  is  found  at  about  160  or  250  mm.  from  the  objective. 

\  14  Numbering  or  Lettering  Objectives. — Instead  of  designating  objectives  by 
their  equivalent  focus,  many  Continental  opticians  use  letters  or  figures  for  this 
purpose.  With  this  method  the  smaller  the  number,  or  the  earlier  in  the  alpha- 
bet the  letter,  the  lower  is  the  power  of  the  objective.  (See  further  in  Ch.  IV,  for 
the  power  or  magnification  of  objectives).  This  method  is  entirely  arbitrary  and 
does  not,  like  the  one  above,  give  direct  information  concerning  the  objective. 

\  15.  Dry  Objectives. — These  are  objectives  in  which  the  space  between  the  front 
of  the  objective  and  the  object  or  cover-glass  is  filled  with  air  (Fig.  22).  Most  ob- 
jectives of  low  and  medium  power  (i.  e.,  J^th  in.  or  3  mm.  and  lower  powers)  are 
dry. 

Fig.  22.  Section  of  a  dry  objective  showing 
working  distance  and  lighting  by  reflected 
light. 

Axis.    The  principal  optic  axis  of  the  ob- 
jective. 

B   C.    Back    Combination,   composed    of  a 
plano-concave   lens   of  flint  glass   (F),    and  a 
double  convex-  lens  of  crown  glass  (c). 
F  C.  Front  Combination. 
C,  O,  si.   The  cover-glass,  object  and  slide. 
Mirror.    The  mirror  is  represented  as  above 
the  stage,  and  as  reflecting  parallel  rays  from 
its  plane  face  upon  the  object. 
Stage.  Section  of  the  stage  of  the  microscope. 

IT.    The  Working  Distance,  that  is  the  distance  from  the  font  of  the  objective  to 
the  object  when  the  objective  is  in  focus. 


•B.C. 


■ 


12 


MICROSCOPE  AND  ACCESSORIES. 


\_CH.  I. 


\  16.  Immersion  Objectives. — An  immersion  objective  is  one  with  which  there 
is  some  liquid  placed  between  the  front  of  the  objective  and  the  object  or  cover- 
glass.  The  most  common  immersion  objectives  are  those  (A)  in  which  water  is 
used  as  the  immersion  fluid,  and  (B)  where  some  liquid  is  used  having  the  same 
refractive  and  dispersive  power  as  the  front  lens  of  the  objective.  Such  a  liquid 
is  called  homogeneous,  as  it  is  optically  homogeneous  with  the  front  glass  of  the 
objective.  It  may  consist  of  thickened  cedar  wood  oil  or  of  glycerin  containing 
some  salt,  as  stannous  chlorid,  in  solution.  When  oil  is  used  as  the  immersion 
fluid  the  objectives  are  frequently  called  oil  immersion  objectives.  The  disturb- 
ing effect  of  the  cover-glass  (Fig.  56)  is  almost  wholly  eliminated  by  the  use  of 
homogeneous  immersion  objectives,  as  the  rays  undergo  very  little  or  no  refraction 
on  passing  from  the  cover-glass  through  the  immersion  medium  and  into  the  ob- 
jective ;  and  when  the  object  is  mounted  in  balsam  there  is  practically  no  refrac- 
tion in  the  ray  from  the  time  it  leaves  the  balsam  till  it  enters  the  objective. 

Fig.  23.  Sectional  view  of  an  Immersion,  Ad- 
justable Objective,  and  the  object  lighted  with 
axial  or  central  and  zvith  oblique  light. 

Axis.    The  principal  optic  axis  of  the  objective. 

B  C,  M  C,  EC.  The  back,  middle  and  front 
combination  of  the  objective,  hi  this  case  the 
front  is  not  a  combination,  but  a  single  piano  con- 
vex-lens. 

A,  B.  Parallel  rays  reflected  by  the  mirror  axi- 
ally  or  centrally  upon  the  object. 

C.  Ray  reflected  to  the  object  obliquely. 

I.  Immersion  fluid  between  the  front  of  the  ob- 
jective and  the  cover-glass  or  object  (O). 

Mirror.   The  mirror  of  the  microscope. 

O.  Object.  It  is  represented  without  a  cover- 
glass.  Ordinarily  objects  are  covered  whether  ex- 
amined zvith  immersion  or  with  dry  objectives. 

Stage.  Section  of  the  stage  of  the  microscope. 

\  17.  Non-Achromatic  Objectives. — These  are  objectives  in  which  the  chro- 
matic aberration  is  not  corrected,  and  the  image  produced  is  bordered  by  colored 
fringes.  They  show  also  spherical  aberration  and  are  used  only  on  very  cheap 
microscopes.     {\\  7,  8,  Figs.  12,  13). 

I  18.  Achromatic  Objectives. — In  these  the  chromatic  and  the  spherical  aberra- 
tion are  both  largely  eliminated  by  combining  concave  and  convex  lenses  of  differ- 
ent kinds  of  glass  "so  disposed  that  their  opposite  aberrations  shall  correct  each 
other."  All  the  better  forms  of  objectives  are  achromatic  and  also  aplanatic  (g  19). 
That  is  the  various  spectral  colors  come  to  the  same  focus. 

\  19.  Aplanatic  Objectives,  etc. — These  are  objectives  or  other  pieces  of  optical 
apparatus  (oculars,  illuminators,  etc.),  in  which  the  spherical  distortion  is  wholly 
or  nearly  eliminated,  and  the  curvatures  are  so  made  that  the  central  and  marginal 
parts  of  the  objective  focus  rays  at  the  same  point  or  level.  Such  pieces  of  appa- 
ratus are  usually  achromatic  also.     (§  7,  8). 

\  20.  Apochromatic  Objectives. —A  term  used  by  Abbe  to  designate  a  form  of 
objective  made  by  combining  new  kinds  of  glass  with  a  natural  mineral  (Calcium 


CI  I.  /.]  MICROSCOPE  AND  ACCESSORIES.  13 

fluoride,  Fluorite,  or  Fluor  spar).  The  name,  Apochroniatic,  is  used  to  indicate 
the  higher  kind  of  achromatism  in  which  rays  of  three  spectral  colors  are  com- 
bined at  one  focus,  instead  of  rays  of  two  colors,  as  in  the  ordinary  achromatic 
objectives.  At  the  present  time  (1896)  several  opticians  make  apochroniatic  ob- 
jectives without  using  the  fluorite.  Some  of  the  apochromatics  deteriorate  rather 
quickly  in  hot,  moist  climates. 

The  special  characteristics  of  these  objectives,  when  used  with  the  "compen- 
sating oculars  "  are  as  follows  : 

(1)  Three  rays  of  different  color  are  brought  to  one  focus,  leaving  a  small  ter- 
tiary spectrum  only,  while  with  objectives  as  formerly  made  from  crown  and  flint 
glass,  only  two  different  colors  could  be  brought  to  the  same  focus. 

(2)  In  the^e  objectives  the  correction  of  the  spherical  aberration  is  obtained  for 
tivj  different  colors  in  the  brightest  part  of  the  spectrum,  and  the  objective  shows 
the  same  degree  of  chromatic  correction  for  the  marginal  as  for  the  central  part  of 
the  aperture.  In  the  old  objectives,  correction  of  the  spherical  aberration  was 
confined  to  rays  of  one  color,  the  correction  being  made  for  the  central  part  of  the 
spectrum,  the  objective  remaining  under  corrected  sphericalh-  for  the  red  rays  and 
ozw-corrtcted  for  the  blue  rays  ($  8). 

(3)  The  optical  and  chemical  foci  are  identical,  and  the  image  formed  by  the 
chemical  rays  is  much  more  perfect  than  with  the  old  objectives,  hence  the  new 
objectives  are  well  adapted  to  photography. 

(4)  These  objectives  admit  of  the  use  of  very  high  oculars,  and  seem  to  be  a 
considerable  improvement  over  those  made  in  the  old  way  with  crown  and  flint 
glass.  According  to  Dippel  (Z.  w.  M.  18S6,  p.  300)  dry  apochroniatic  objectives 
give  as  clear  images  as  the  same  power  water  immersion  objectives  of  the  old  form. 

\  21.  Non-Adjustable  or  Una.ijustable  Objectives. — Objectives  in  which  the 
lenses  or  lens  systems  are  permanently  fixed  in  their  mounting  so  that  their  rela- 
tive position  always  remains  the  same.  Low  power  objectives  and  those  with 
homogeneous  immersion  are  mostly  non-adjustable.  For  beginners  and  those  un- 
skilled in  manipulating  adjustable  (§  22)  objectives,  non-adjustable  ones  are  more 
satisfactory,  as  the  optician  has  put  the  lenses  in  such  a  position  that  the  most 
satisfactory  results  may  be  obtained  when  the  proper  thickness  of  cover-glass  and 
tube-length  are  employed.  (See  table  of  tube-length  and  thickness  of  cover-glass 
below  Fig.  24). 

\  22.  Adjustable  Objectives.— An  adjustable  objective  is  one  in  which  the  dis- 
tance between  the  systems  of  lenses  (usually  the  front  and  the  back  systems)  may 
be  changed  by  the  observer  at  pleasure.  The  object  of  this  adjustment  is  to  cor- 
rect or  compensate  for  the  displacement  of  the  rays  of  light  produced  by  the 
mounting  medium  and  the  cover-glass  after  the  rays  have  left  the  object.  It  is 
also  to  compensate  for  variations  in  "tube-length."  See  \  24.  As  the  displace- 
ment of  the  rays  by  the  cover-glass  is  the  most  constant  and  important,  these  ob- 
jectives are  usually  designated  as  having  cover-glass  adjustment  or  correction. 
(Fig.  23.     See  also  practical  work  with  adjustable  objectives  \  96). 

\  23.  Parachromatic,  Pantachromatic  and  Semi-apochromatic  Objectives. — These 
are  trade  names  for  objectives,  most  of  them  containing  one  or  more  lenses  >>f  the 
new  Jena  glass.  They  are  said  to  approximate  much  more  closely  to  the  apo- 
chromatics than  to  the  ordinary  objectives. 

\  24.  Variable  Objective. — This  is  a  low  power  objective  of  36  to  26  111111.  equi- 
valent focus,  depending  upon  the  position  of  the  combinations.     By   means  of  a 


14  MICROSCOPE  AND  ACCESSORIES.  {CH.  I. 

screw  collar  the  combinations  may   be  separated,  diminishing  the  power  or  ap- 
proximated and  thereby  increasing  it. 

\  25.  Projection  Objectives. — Tliese  are  designed  especially  for  projecting  an 
image  on  a  screen  and  for  photo- micrography.  They  are  characterized  by  having  a 
fiat,  sharp  field  brilliantly  lighted.  In  power  they  vary,  the  lowest  being  of  75 
mm.  and  the  highest  of  6  mm.  equivalent  focus  (see  Ch.  IV). 

\  26.  Illuminating  or  Vertical  Illuminating  Objectives. — Tbese  are  designed  for 
the  stud}-  of  opaque  objects  with  good  reflecting  surfaces,  like  the  rulings  on  metal 
bars.  Tbe  light  enters  the  side  of  the  tube  or  objective  and  is  reflected  vertically 
downward  through  the  objective  and  thereby  is  concentrated  upon  the  object.  The 
object  reflects  part  of  the  light  back  into  the  microscope  thus  enabling  one  to  see 
a  clear  image. 

\  27.  Tube-Length  and  Thickness  of  Cover-Glasses.  —  "In  the  construction  of 
microscopic  objectives,  the  corrections  must  be  made  for  the  formation  of  the 
image  at  a  definite  distance,  or  in  other  words  the  tube  of  the  microscope  on 
which  the  objective  is  to  be  used  must  have  a  definite  length.  Consequently  the 
microscopist  must  know  and  use  this  distance  or  '  microscopical  tube-length  '  to 
obtain  the  best  results  in  using  an}-  objective  in  practical  work."  Unfortunately 
different  opticians  have  selected  different  tube-lengths  and  also  different  points 
between  which  the  distance  is  measured,  so  that  one  must  know  what  is  meant  by 
the  tube-length  of  each  optician  whose  objectives  are  used.     See  table. 

The  thickness  of  cover-glass  used  on  an  object  (see  Ch.  VII,  on  mounting),  ex- 
cept with  homogeneous  immersion  objectives,  has  a  marked  effect  on  the  light 
passing  from  the  object  ( Fig.  56).  To  compensate  for  this  the  relative  positions  of 
the  systems  composing  the  objective  are  different  from  what  they  would  be  if  the 
object  were  uncovered.  Consequently,  in  non -adjustable  objectives  some  standard 
thickness  of  cover-glass  is  chosen  by  each  optician  and  the  position  of  the  systems 
arranged  accordingly.  With  such  an  objective  the  image  of  an  uncovered  object 
would  be  less  distinct  than  a  covered  one,  and  the  same  result  would  follow  the  use 
of  a  cover-glass  much  too  thick. 


CII.  /.] 


MICROSCOPE  AND  ACCESSORIES. 


<5 


Tube  Length  "  by 


rube-lengtb     in 
Millimeti 


Length  in  Millimeters  and  Raits  included  in 

Various  Opticians* 

rts.  included 
in  "  Tube- 
length." 

Sec  Diagram. 

Gruuow,  New  York 203111m. 

E.  Leitz,  Wetzlar       170  mm. 

Nachet  et  Fils,  Paris  .    .    •    • i.|6  or  200  111111. 

Powell  and  Zealand,  London  ....       254  mm. 

C.  Reichert,   Vienna 160  to  180  nun. 

Spencer  Lens  Co.,  Buffalo 235  or  160  nun. 

\W.  Wales,  New  York 254  mm. 

Bausch  &  Loinb  Opt.  Co.,  Rochester.  .  216  or  160  mm. 

Rezu,  Hausser  et  Cie,  Paris 220  mm. 

!  Klonne  und  Miiller,  Berlin i6o-i8oor  254  mm. 

)  W.  &  H.  Seibert,  Wetzlar 190  mm. 

I  Swift  &  Son,  London 165  to  228^  mm. 

[  C.  Zeiss,  Jena 160  or  250  mm. 

Gundlach  Optical  Co.,  Rochester  .    .    .  254  mm. 

R.  Winkel,  Gottingen 220  mm. 

Ross  &  Co. ,  Loudon 254111m. 

R.  &  J.  Beck,  London 254  mm. 

J.  Green,  Brooklyn 254  mm. 

Hartnack,  Potsdam 160-1S0  mm. 

VeVick  (Stiassnie)  Paris 160-200111111. 

Watson  &  Sons,  London 160-250  mm. 

Thickness  of  Cover  Glass  for  Which  Non- Adjustable  Objectives  are  Corrected  by 

Various  Opticians. 

[" J.  Green,  Brooklyn, 
j  J.  Gruuow,  Brooklyn. 
I  -  ,  mrn.  |  Powell  and  Lealand,  London. 

I  Spencer  Lens  Co.,  Buffalo. 
[  W.  Wales,  New  York. 
Watson  &  Sous,  London. 
Klonne  und  Miiller,  Berlin. 
j  E.  Leitz,  Wetzlar. 
I  R.  Winkel,  Gottingen,  German}'. 
Ross  &  Co.,  London. 
Bausch  &  Lomb  Optical  Co.,  Rochester. 
';■,;,' 'mm.  C.  Zeiss,  Jena. 

',, ,,''111111.  C.  Reichert,  Vienna. 

!  Gundlach  Optical  Co.,  Rochester. 
W.  &  H.  Seibert,  Wetzlar. 
R.  &J.  Beck,  London, 
mm.  J.  Zentmayer,  Philadelphia. 

Nachet  et  Fils,  Paris. 
Bezu,  Hausser  et  Cie,  Paris. 
Swift  and  Son,  London. 


Fig.  24. 


rVirmm- 
Ajfomm. 

Jj/jjUim. 

mm. 
/'.mm. 

15      2  1 


12      I  ' 
"I  (Ml 

in       1    ■. 
Inn 


I  n 

TOO 


mm. 


*The  information  contained  in  these  tables  was  very  kindly  furnished  by  tin- 
opticians  named,  or  by  consulting  catalogs.  In  most  of  the  later  catalogs  the  in- 
formation is  definite,  and  many  makers  now  r.ot  only  put  their  names  and  the 
equivalent  focal  length  on  their  objectives,  but  they  ;'.<ld  the  numerical  aperture 
(I  29)  and  the  tube-length  for  which  the  objective  is  corrected.  This  is  in  accord- 
ance with  the  recommendations  of  the  author  in  the  original  paper  on  'tube- 
length,"  (Proc.  Amer.  Soc   Micr.,   Vol.   IX,  p.   168.  also  by   Bausch,  Vol.  XII,  p. 


i6 


MICROSCOPE  AND  ACCESSORIES. 


[CH.  I. 


$  28.  Aperture  of  Objectives. — The  angular  aperture  or  angle  of 
aperture  of  an  objective  is  the  angle  "  contained,  in  each  case,  between 
the  most  diverging  of  the  rays  issuing  from  the  axial  point  of  an  object 
[/.  <\,  a  point  in  the  object  situated  on  the  extended  optic  axis  of  the 
microscope]  ,  that  can  enter  the  objective  and  take  part  in  the  formation 
of  an  image."      (Carpenter). 

43).  If  the  table  in  this  edition  is  compared  with  the  original  table  or  with  that  in 
the  previous  edition  of  this  book  some  differences  will  be  noted,  the  changes  being 
in  the  direction  of  uniformity  and  in  general  in  the  direction  recommended  by  the 
writer  and  Mr.  Bausch  and  the  committee  of  the  American  Microscopical  Society. 
The  recommendations  of  the  committee,  published  in  the  Proceedings,  Vol.  XII, 
p.  250,  are  as  follows  : 

"  Believing  in  the  desirability  of  a  uniform  tube-length  for  microscopes,  we 
unanimously  recommend  :  1.  That  the  parts  of  the  microscope  included  in  the 
tube-length  should  be  the  same  by  all  opticians,  and  that  the  parts  included  should 
be  those  between  the  upper  end  of  the  tube  where  the  ocular  is  inserted  and  the 
lower  end  of  the  tube  where  the  objective  is  inserted. 

2.  That  the  actual  extent  of  tube  length 
as  defined  in  section  r  —  Be,  for  the  short 
or  continental  tube,  160  mm.,  or  6.3  inch- 
es, and  216  mm.,  or  8)4  inches,  for  the 
long  tube,  and  that  the  draw  tube  of  the 
microscope  possess  two  special  marks  in- 
dicating these  standard  lengths. 

3.  That  oculars  be  made  par-focal,  and 
that  the  par-focal  plane  be  coincident 
with  that  of  the  upper  end  of  the  tube. 

4.  That  the  mounting  of  all  objectives 
of  6  mm.  {%  inch)  and  shorter  focus 
should  be  such  as  to  bring  the  optical 
center  of  the  objective  1%  inches  below 
the  shoulder,  and  that  all  objectives  be 
marked  with  the  tube-length  for  which 
they  are  corrected. 

5.  That  non-adjustable  objectives  be 
corrected  for  cover-glass  from  T\f5  to  ^ 
mm.  (xf<r  to  t|q  inch)  in  thickness. 

These  recommendations  give  a  distance 

Fig.  25.    The  tube  of  a  microscope  with    0f  10  inches  (254  mm.)  between  the  par- 

ocular  micrometer  and  nose  piece  in    focai  piane  Qf  the  ocular  and  the  optical 

position   to  show  that  in  measuring   center  of  the  objective  for  the  long  tube, 

tube-length  one  must  measure  from    an(j   are   essentially   in  accord  with   the 

the  eye  lens  to  the  place  where  the  ob-   actUal  practice  of  opticians. 

jective  is  attached.     {Zeiss'  Catalog,        At  the  request  of  the  committee,  a  joint 

No.  30).  conference  was  held  with  the   opticians 

belonging  to  the  Society  and  present  at  the  meeting.     They  expressed  their  belief 

in  the  entire  practicability  of  the  above  recommendations  and  a  willingness  to 

adopt  them." 

(Signed)  Simon  H.  GaGE, 

A.  Clifford  Mercer, 
Charles  E.  Barr. 


<  v/.  /.  i  Mh  a\  >s<  ■< >/•/■;  AND  .  i'  v  7-;.ssv >ava.v. 

In  general,  the  angle  increases  with  the  size  of  the  lens<  s  forming  the  obj<  i 
and  the  shortness  of  the  equivalent  focal  distanci  If  all  objecti 

dry  or  all  water  or  all  homogeneous  immersion  a  comparison  of  the  angulai 
ture  would  l;  i  \  <. •  one  a  g 1  idea  of  the  relative  number  of  image  formi 

I'n,.  26.  Diagram  illustrating  the  angular  aperture  of  a 
microscopic  objective.  Only  the  front  lens  of  the  objet  tive  is 
shown. 

.  I  tris,  the  principal  optic  axis  of  the  objectiv 

/:.  I .  /.'  ( \  the  most  divergent  rays  that  can  enter  the  objective, 
they  mark  the  angular  aperture.  .  /  B  />  or  < '  />'  />  half  the 
angular  aperture.  This  is  designated  by  11  in  making  Vw- 
merical  .  Aperture  computations.     Sec  the  tabic.  $  30. 

transmitted  by  different  objectives;  but  as  some  are  dry, 
others  water  and  still  others  homogeneous  immersion,  one 
ran  sit  at  a  glance  that,  other  things  being  equal,  the 
dry  objective  (Fig.  27)  receives  less  light  than  the  water  immersion,  and  tin 
water  immersion  (Pig.  28)  less  than  iIk-  homogeneous  immersion  (Fig.  29). 
In  order  to  render  comparison  accurate  between  different  kinds  of  objectiv< 
Professor  Abbe  takes  into  consideration  the  rays  actually  passing  from  the  hack 
combination  of  the  objective  to  form  the  real  image  ;  he  thus  takes  into  account 
the  medium  in  front  of  the  objective  as  well  as  the  angular  aperture.  The  term 
"  Numerical  .  \perture"  1  N.  .  1 .  1  was  introduced  by  Abbe  to  indicate  the  capacity 
of  an  optical  instrument  "  for  receiving  rays  from  the  object  and  transmitting 
them  tu  the  image. 

\  29.  Numerical  Aperture  (abbreviated  N.  A.),  as  now  employed  for  micro- 
scope objectives,  is  the  ratio  of  the  semi-diameter  of  the  emergent  pencil  to  the 
focal  length  of  the  lens.  Or  as  the  factors  are  more  readily  obtainable  it  is 
simpler  to  utilize  the  relationship  shown  in  the  I, a  Grange-Helmholtz  formula, 
and  indicate  the  aperture  by  the  expression  :  N.  A.  n  sin  11.  In  this  formula  ;/ 
is  the  index  of  refraction  of  the  medium  in  front  of  the  objective  (air,  water  or 
homogeneous  liquid  >,  and  sin  11  is  the  sine  of  half  the  angle  of  ap<  rtur<  Fig.  26, 
1)    B   A).       For    the    mathematical    discussion    showing     that    the     expressions 

semi-diameter  of  emergent  pencil  .  ,  , 

.       ,  ,         ,      .  ,     ,  =11  sm  u,  the  student  is  referred  to  the     oiinial 

Focal  length  ot  the  lens 

of  the  Royal  Microscopical  Society \  r88i,  pp.  392-395. 

For  example,  take  three  objectives  each  of  3  mm.  equivalent  focus,  one  being  a 
dry.  one  a  water  immersion,  and  one  a  homogeneous  immersion.  Suppose  that 
the  dry  objective  has  an  angular  aperture  of  ro6°,  the  water  immersion  of  94  and 
the  homogeneous  immersion  of  900.  Simply  compared  as  to  their  angular  aper- 
ture, without  regard  to  the  medium  in  front  of  the  objective,  it  would  look  as  if 
the  dry  objective  would  actually  take  in  and  transmit  a  wider  pencil  of  light  than 
either  of  the  others.  However,  if  the  medium  in  front  of  the  objective  is  con- 
sidered, that  is  to  saw  if  the  numerical  instead  of  the  angular  apertures  are  com- 
pared, the  results  would  he  as  follows:  Numerical  Aperture  of  a  dry  objective  of 
[060,  N.  A.  //sin  11.  In  the  case  of  dry  objectives  the  medium  in  front  of  the 
objective  being  air,  the  index  of  refraction  is  unity,  whence  //  1.  Half  the 
angular  aperture  is  '"'°  530.  By  consulting  a  table  of  natural  sines  it  will 
found  that  the  sine  of  530  is 0.799,  whence  X.  A.       //  or  1  x  sin  //  or  1  >. 

*  \  29a.  Interpolation.  In  practice,  as  in  solving  problems  similar  to  those  on 
the  following  pag<  s  and  those  in  n  fraction  on  p.  50,  if  one  cannot  find  a  sine  exactly 


[8 


MICROSCOPE  AND  ACCESSORIES. 


\CH.  I. 


c —       - 

_omP> 

^-^ 

^\\ 

/AC 

WW 

1  ///Cort  * 

W^ 

-^- —      tj^ 
*~~ — 

JoM> 

\V 

;    '   /    $ 

\\\\\ 

J  / J f/C  o  ire  r 

the  angular  aperture  is 


I  n.s.  27-29  are  somewhat  modified  from 
Ellenberger,  and  air  introduced  to  illustrate 
the  relative  amount  of  utilized  light,  ivith  dry, 
water  immersion  and  homogeneous  immer- 

27  sion  objectives  of  the  same  equivalent  focus. 
The  point  from  which  the  rays  emanate  is  in 
air  in  each  case.  If  Canada  balsam  :vere  in 
place  of  the  air  beneath  the  cover  glass  there 
would  he  practically  no  refraction  of  the 
rays  on  entering  the  cover  glass  ( ',.  /6). 

FiG.  27.  Showing  the  course  of  the  rays 
passing  through  a  cover  glass  from  an  axial 
point   of  the   object,    and   the    number   that 

28  'finally  enter  the  front  of  a  dry  objective. 

Fig.  28.  Rays  from  the  axial  point  of  the 
object  traversing  a  cover  of  the  same  thick- 
ness as  in  Fig.  27,  and  entering  the  front 
tens  of  a  water  immersion  objective. 

FiG.  29.  Rays  from  an  axial  point  of  the 

29  object  traversing  a  cover  glass  and  entering 
the  front  of  a  homogeneous  immersion 
object  rve. 

With  the  water  immersion  objective  the 
medium  in  front  is  water,  audits  index  of 
refraction  is  1.33,  whence?/  =  1.33.      Half 
-V°  =  470,  and  by  the  table  the  sine  of  470  is 


corresponding  to  a  given  angle  ;  or  if  one  has  an  angle  which  does  not  correspond 
to  an\-  sine  or  angle  given  in  the  table,  the  sine  or  angle  may  be  closely  approxi- 
mated by  the  method  of  interpolation,  as  follows  :  Find  the  sine  in  the  table  nearest 
the  sine  whose  angle  is  to  be  determined.  Get  the  difference  of  the  sines  of  the  an- 
gles greater  and  less  than  the  sine  whose  angle  is  to  be  determined.  That  will  give 
the  increase  of  sine  for  that  region  of  the  arc  for  15  minutes.  Divide  this  increase  by 
15  and  it  will  give  with  approximate  accuracy  the  increase  for  1  minute.  Now 
get  the  difference  between  the  sine  whose  angle  is  to  be  determined  and  the  sine 
just  below  it  in  value.  Divide  this  difference  by  the  amount  found  necessary  for 
an  increase  in  angle  of  1  minute  and  the  quotient  will  give  the  number  of  minutes 
greater  the  sine  is  than  the  next  lower  one  whose  angle  is  known.  Add  this  num- 
ber of  minutes  to  the  angle  of  the  next  lower  sine  and  the  sum  will  represent  the 
desired  angle  of  the  sine.  Or  if  the  sine  whose  angle  is  to  be  found  is  nearer  in 
size  to  the  sine  just  greater,  proceed  exactly  as  before,  getting  the  difference  in  the 
Hues,  but  subtract  tin-  number  of  minutes  of  difference  and  the  result  will  give  the 
angle  sought.  For  example  take  the  case  in  the  last  section  where  the  sine  of  the 
angle  of  280  54/  is  given  as  0.4S327.  If  one  consults  the  table  the  nearest  sines 
found  are  0.48099,  the  sine  of  280  45',  and  0.48481,  the  sine  of  290.  KvidentW 
then  the  angle  sought  must  lie  between  280  45'  and  290.  If  the  difference  between 
o.  J S48 1  and  0.48099  l»e  obtained,  0.48481  — 0.48099  =0.00382,  and  this  increase  for 
15'  be  divided  by  15  it  will  give  the  increase  for  1  minute  ;  0.00382-7-15  =  0.000254. 
Now  the  difference  between  the  sine  whose  angle  is  to  be  found  and  the  next 
lower  sine  is  0.48327  —  0.48099  =  0.00228.  If  this  difference  is  divided  by  the 
amount  found  necessary  for  1  minute  it  will  give  the  total  minutes  above  2S0  45'  ; 
0.00228  -=-  0.000254  =  9.  That  is,  the  angle  sought  is  9  minutes  greater  than 
280  45' =  28°  54'. 


CH.  /.]  MICROSCOPE  AND  ACCESSORIES. 

found  to  be  0.731,  /.  e.,  sin  11  =  0.731,  whence  N.  A.  =  //or  1.33  x  sin  u 
or  0.731  =  0.972. 

With  thf  oil  immersion  in  the  same  way  N.  A.  =  //  sin  //  ;  //  or  the 

index  of  refraction  of  the  homogeneous  fluid  in  front  of  th< 

1.52,  and  the  semi-angle  of  aperture  is  '\"°  =  450.      The  sine  of  4.5 

0.707,  whence  N.  A.  =  n  or  1.52  X  sin  u  or  0.707  =  1.074. 

By  comparing  these   numerical   apertures:   Dry  0.799,   water  o.g 
homogeneous  immersion  1.074,  the  same  idea  of  the  real  light  efficiency 
and  image  power  of  the  different  objectives  is  obtained,  as  in  th  »hic 

representations  shown  in  Figs.  27-29. 

If  one  knows  the  numerical  aperture  (N.  A.)  of  an  objective  the  an- 
gular aperture  is  readily  determined  from  the  formula  ;  and  one  can  de- 
termine the  equivalent  angles  of  objectives  used  in  different  media  I  /.  e. , 
dry  or  immersion).  For  example,  suppose  each  of  three  objectives  has 
a  numerical  aperture  (N.  A.)  of  0.80,  what  is  the  angular  aperture  of 
each.  Using  the  formula  of  N.  A.  =  n  sin  u  one  has  N.  A.  =  0.80  for 
all  objectives. 
For  the  dry  objective  n  =  1  (Refractive  index  of  air). 

"        water  immersion  objective  n  =  1.33  (Refractive  index  of  water), 
homogeneous  immersion  objective  n  =  1.52   (Refractive  index 
of  homogeneous  liquid).     And  2  it  is  to  be  found  in  each  case. 

For  the  dry  objective,  substituting  the  known  values  the  formula 
becomes  0.80  =  1  sin  u,  or  sin  it  =  0.80.  By  inspecting  the  table  of 
natural  sines  (3d  page  of  cover)  it  will  be  found  that  0.80  is  the  sine  of 
53  degrees  and  8  minutes.  As  this  is  half  the  angle  the  entire  angular 
aperture  of  the  dry  objective  must  be  530  8'  x  2  =  1060  16'. 

For  the  water  immersion  objective,  substituting  the  known  values 

in  the  formula  as  before  :  0.80  =  1.33  sin  it,  or  sin  it  =  =  0.601  s. 

*-33 
Consulting  the  table  of  sines  as  before,  it  will  be  found  that  0.6015  's  the 

sine  of  360  59'  whence  the  angular  aperture  (water  angle)  is  360  59' 
X  2  =  730  58'. 

For  the  homogeneous  immersion  objective,  substituting  the  known 
values,    the    formula    becomes  :    0.80  =  1.52    sin    u   whence   sin  <v  = 

=  0.5263.     And  by  consulting  the  table  of  sines  it  will  be  found 
*  o 

that  this  is  the  sine  of  310  45  J  -J'  whence  2  it  or  the  entire  angle  balsam 
or  oil  angle)  is  630  31'. 

That  is,  three  objectives  of  equal  resolving  powers,  each  with  a  nu- 
merical aperture  of  0.80  would  have  an  angular  aperture  of  [06  16'  in 
an">  73°  58'  in  water,  and  630  31'  in  homogeneous  immersion  liquid. 

For  the  apparatus  and  method  of  determining  aperture,  see  appendix. 


20 


MICROSCOPE  AND  ACCESSORIES. 


{CH.  I. 


§  t,o.  Table  of  a  Group  of  Objectives  with  the  Numerical  Aper- 
ture (N.  A.)  and  the  method  of  obtaining  it.  Half  the  angular  aper- 
ture is  designated  by  u  and  the  index  of  refraction  of  the  medium  in  front 
of  the  objective  by  n.  For  dry  objectives  this  is  air  and  n  =  i ,  for  water 
immersions  n  =  f.JJ,  and  for  homogeneous  immersions  n  =  1.52.  {For 
a  table  of  natural  sines,  see  third  page  of  cover). 


(<3^      Natural  Sine 
Objective.     "3  "S  2    of  half  the  anSular 

;  be  &!-_'  I  aperture. 

=  <  (sinu). 


25  mm. 

(Dry.) 

25  mm. 

(Dry.) 

j2}4  mm. 
(Dry.) 

12)4  mm. 
(Dry.) 


20u 


40" 


42c 


IOO" 


6  mm.  c 

(Dry.) 


6  mm. 
(Dry.) 

3  mm. 
(Dry.) 

3  mm. 
(Dry.) 

2  mm. 

Water. 

Immersion. 

2  mm. 


136° 
1150 
163° 


Sin  — =0.1736 
2 

Sin  4°  =  0.3420 


Sin  i2  =0.3583 
2 

Sin  ^  =  0.7660 
2 

Sin-5  =  06087 
2 

Sin  ="_  =  0.9272 
2 

Sin  H5  =  0.8434 
2 

Sin -^  =  0.9890 
2 


Index  of 

Refraction  j     NUMERICAL  APERTURE 
of  the  medi-! 

urn  in  front!  .  . 

oftheobjec-j  (N.A.  )  =  7J  Sill  tt. 

tive.  («). 


»=l        N.A.  =  1X0.1736  =  0.173 

#  =  1       N.A.  =  1X0.3420  =  0.342 

N.A.  =  1  xo.35S3  =  o.35S 

N.  A.  =  1  X  o.  7660  =  o.  766 

N.A.  =  1X0.6087  =  0.608 

N.A.  =  1X0.9272=0.927 

N.A.  =  1  X  0.8434  — 0.843 

n  =  i       N.A.  =  1X0.9890  =  0.989 


n  =  1 


n  =  1 


;/       1 


;/  -     1 


n  =  1 


96°i2r  j  Sin  ^  =074431     »==i.33|N.A.  =  1.33X0.7443  =  0.99 

N.A.  =  1.52  X0.8223  =  1.25 


'         no°  ^8' 
Homogeneous  iio°38/  Sin =-=0.8223;     n  =  1.52 

Immersion. 


2  mm. 

Homogeneous 

Immersion. 


1 340 io' 


Sin 


i34°ic/_ 


:0.92I0 


n  =  1.52 


N.A.  =  1.52  X0.9210  =  1.40 


§  31.  Significance  of  Aperture. — As  to  the  real  significance  of 
aperture  in  microscopic  objectives,  it  is  now  an  accepted  doctrine  that — 
the  corrections  in  spherical  and  chromatic  aberration  baiug  the  same — 
(1)  Objectives  vary  directly  as  their  numerical  aperture  in  their  ability 
to  define  or  make  clearly  visible  minute  details  (resolving  power).  For 
example  an  objective  of  4  mm.  equivalent  focus  and  a  numerical  aper- 
ture of  0.50  N.  A.  would  define  or  resolve  only  half  as  many  lines  to 


CH.  /.]  MICROSCOPE  AND  ACCESSORIES.  21 

the  millimeter  or  inch  as  a  similar  objective  of  i.oo  X.  A.     So  also  an 

objective  of  2  mm.  focus  and   1.40   X.  A.  would  resolve  only  t\\: 
many  lines  to  the  millimeter  as  a  4  mm.  objective  of  >>.-■>  X.  A.     Thus 
it  is  seen  that  defining  power  is  not  a  result  of  magnification  but 
aperture,  otherwise  the   2   mm.  objective  would   resolve   far  more  than 
twice  as  many  lines  as  the  4  mm.  objective. 

(2)  The  illuminating  power  of  an  objective  of  a  given  focus  is  found 
to  vary  directly  as  the  square  of  the  numerical  aperture  1  X.  A.  J.  Thus 
if  two  4  mm.  objectives  of  X.  A.  0.20  and  X.  A.  0.40  were  compared 
as  to  their  illuminating  power  it  would  be  found  from  the  above  that 
the}'  would  vary  as  0.20*  :  0.40'''=  0.0400  :  o.  1600  or  1  :  4.  That  is  the 
objective  of  0.20  N.  A.  wotdd  have  but  j^th  the  illuminating  power  of 
the  one  of  0.40  N.  A. 

In  considering  illuminating  power  the  equivalent  focal  length  must 
also  be  considered.  If  the  N.  A.  were  the  same  in  a  3  mm.  and  a  6 
mm.  objective  their  illuminating  power  would  vary  directly  with  the 
square  of  the  foci.  Thus  the  illuminating  power  of  the  6  and  the  3 
mm.  objectives  would  be  as  62  :  3s  or  36  to  9  or  4  :  1,  that  is  4  times  as 
great  in  the  6  mm.  as  in  the  3  mm.  objective.  As  the  magnification  of 
an  objective  varies  indirectly  as  the  equivalent  focus,  it  follows  also 
that  the  illuminating  power  will  vary  indirectly  as  the  square  of  the 
magnification  of  the  objective.  The  magnification  of  the  6  mm.  is  42 
and  of  the  3  mm.  84,  whence  the  illuminating  power  of  the  two  objec- 
tives are  as  422  :  84"  or  1764  :  7056  or  1  :  4.  As  the  ratio  is  inverse  in 
this  case  the  result  is  the  same  as  before,  that  is  4  times  as  great  for  the 
6  mm.  as  for  the  3  mm.  objective. 

(3)  The  penetrating  power,  that  is  the  power  to  see  more  than  one 

plane,  is  found  to  vary  as  the  reciprocal  of  the  numerical  aperture 

X.  A. 

so  that  in  an  objective  of  a  given  focus  the  greater  the  aperture  the  1 

the  penetrating  power. 

In  comparing  the  penetrating  power  of  objectives  of  different  foci, 
the  numerical  aperture  being  the  same,  it  is  found  that  the  penetrating 
power  increases  directly  as  the  square  of  the  focus.  For  example,  two 
objectives  of  the  same  N.  A.,  one  of  4  mm.  and  the  other  of  2  mm. 
focus,  the  penetrating  power  would  be  as  41  :  21  or  10  :  4  or  4  :  1.  That 
is,  the  numerical  aperture  remaining  the  same,  the  greater  the  equiva- 
lent focus  the  greater  the  penetration. 

To  briefly  summarize:  The  numerical  aperture  is  concerned  with 
resolution  and  the  resolving  power  varies  directly  as  the  numerical 
aperture,  thus  if  the  X.  A.  is  doubled,  twice  as  many  lines  to  the  mil- 
limeter or  inch  can  be  resolved. 


22 


MICROSCOPE  AND  ACCESSORIES. 


[CH.  I. 


With  illuminating  and  penetrating  power  the  equivalent  focus  of  the 
objective  must  be  considered  as  well  as  the  numerical  aperture.  With 
objectives  of  the  same  equivalent  focus,  to  double  the  N.  A.  is  to  in- 
crease the  illuminating  power  4-fold  but  the  penetrating  power  is  halved. 

The  numerical  aperture  remaining  constant,  the  illuminating  and 
penetrating  power  vary  directly  as  the  square  of  the  equivalent  focus  ; 
thus  a  4  mm.  objective  would  give  four  times  the  illuminating  and 
penetrating  power  of  a  2  mm.  objective. 

Of  course  when  equivalent  focus  and  numerical  aperture  both  differ 
the  problem  becomes  more  complex. 

For  a  consideration  of  the  aperture  question,  its  history  and  signifi- 
cance, see  J.  D.  Cox,  Proc.  Amer.  Micr.  Soc,  1884,  pp.  5-39;  Jour. 
Roy.  Micr.  Soc,  1881,  pp.  303,  348,  365,  38S  ;  1882,  pp.  300,460; 
1883,  p.  790  ;   1884,  p.  20.     Carpenter-Dallinger,  Chapters  II  and  V. 

THE    OCULAR. 


\  32  A  Microscopic  Ocular  or  Eye-Piece  consists  of  one  or  more  converging 
lenses  or  lens  systems,  the  combined  action  of  which  is,  like  that  of  a  simple  mi- 
croscope, to  magnify  the  real  image  formed  by  the  objective. 

Fig.  30.  Sectional  view  of  a  Huygenian  ocular  (Hg.  ocu- 
lar*), to  show  the  formation  of  the  Eye-Point. 

Axis.  Optic  axis  of  the  ocular.  D.  Diaphragm  of  the 
ocular.     E.  L.  Eye-Lens.     F.  L.  Field-Lens. 

E.  P.  Eye-point.  As  seen  in  section,  it  appears  something- 
like  an  hour-glass.  When  seen  as  in  looking  into  the  ocu- 
lar, i.  e.,  in  transection,  it  appears  as  a  circle  of  light.  It  is 
at  the  point  'where  the  most  rays  cross. 

Depending  upon  the  relation  and  action  of  the  different 
lenses  forming  oculars,  they  are  divided  into  two  great  groups, 
negative  and  positive. 

\  33.   Negative  Oculars  are  those  in  which  the  real,    in- 
verted image  is  formed  within  the  ocular,  the  lower  or  field- 
lens  serving  to  collect  the  image-forming  rays  somewhat,  so 
that  the  real  image  is  smaller  than  as  if  the  field-lens  were 
absent  (Fig.  21).     As  the  field-lens  of  the  ocular  aids  in  the 
formation  of  the  real  image  it  is  considered  by  some  to  form 
a  part  of  the  objective  rather  than  of  the  ocular.     The  upper 
or  eye  lens  of  the  ocular  magnifies  the  real  image. 
\  34.   Positive  Oculars  are  those  in  which  the  real,  inverted  image  of  the  objec- 
tive is  formed  outside  the  ocular,  and  the  entire  system  of  ocular  lenses  magnifies 
the  real  image  like  a  simple  microscope  (Fig.  16). 

Positive  and  negative  oculars  may  be  readily  distinguished,  as  a  positive  ocular 
may  be  used  as  a  simple  microscope,  while  a  negative  ocular  cannot  be  so  used 
when  its  field  lens  is  in  the  natural  position  toward  the  object.     By  turning  the 


CH.  1. 1  MlCROS(  <  >  1  •/:  AND  ACl  ESSOR I ES. 

eye-lens  toward  the  object  and  looking  into  the  field  lens  an  imam-  may  be  seen, 
however. 

Special  names  have  also  been  applied  to  oculars,  depending  upon  tin-  designer, 
the  construction,  or  the  special  use  to  which  the  ocular  is  to  he  applied.  The  fol- 
lowing are  most  used. 

*  In  works  and  catalogs  concerning  the  microscope  and  microscopic  '.us, 

and  in  articles  upon  the  microscope  in  periodicals,  various  forms  of  oculars  or  « 
pieces  are  so  frequently  mentioned,   without   explanation  or  definition,   that  it 
seemed  worth  while  to  give  a  list,  with  the  French  and  German  equivalents,  and  a 

brief  statement  of  their  character. 

Achromatic  Ocular;  Fr.   Oculaire  achromatique ;  Ger.  achromatisches  Okular. 
Oculars  in  which  chromatic  aberration  is  wholly  or  nearly  eliminated.     Aplanatic 
Ocular  ;  Fr.  Oculaire  aplanatique  ;  Ger.  aplanatisches  Okular  (see  \  191.     Bin 
tar,  stereoscopic  Ocular  ;  Fr.  Oculaire  binoculaire  stereoscopique  ;  Ger.  stereosko- 
pisches  Doppel- Okular.     An  ocular  consisting  of  two  oculars  about  as  far  apart  as 
the  two  eyes.     These  are  connected  with  a  single  tube  which  fits  a  monocular  mi- 
croscope.    By  an  arrangement  of  prisms  the  image  forming  rays  are  divided,  half 
being  sent  to  each  eye.     The  most  satisfactory  form  was  worked  out  by  Tolles  and 
is  constructed  on  true  stereotomic  principles,  both  fields  being  equally  illuminated. 
His  ocular  is  also  erecting.     Campani' s  Ocular   (see   Huygenian    Ocular).     Com- 
pound  Ocular  ;   Fr.  Oculaire  compose  ;  Ger.  zusammengesetztes  Okular.     An  ocu- 
lar of  two  or  more  lenses,  e.  g.,  the  Huygenian  (see  F"ig.  30).     Continental  Ocular. 
An  ocular  mounted  in  a  tube  of  uniform   diameter  as  in   Fig.  31.     Deep  Ocular, 
see  high  ocular.     Erecting  Ocular  ;   Fr.   Oculaire  redresseur  ;  Ger.   bildumkeh- 
rendes  Okular.     An  ocular  with  which  an  erecting  prism  is  connected  so  that  the 
image  is  erect  as  with  the  simple  microscope.     Such  oculars  are  most  common  on 
dissecting   microscopes.     Filar  micrometer  Ocular;   Screw  m.  o.   Cobweb  m.  o. 
Ger.  Okular-Schraubenmikrometer.     A  modification  of  Ratnsden's  Telescopic  Cob- 
web micrometer  ocular.     Goniometer  Ocular;  Fr.  Oculaire  a  gouiometre  ;  Ger. 
Goniometer-Okular.     An  ocular  with  goniometer  for  measuring  the  angles  of  mi- 
nute crystals.     High  Ocular,  sometimes  called  a  deep  ocular.     One  that  magnifies 
the  real  image  considerably,  /.  c,  10  to  20  fold.     Huygenian  Ocular,  rluygeus'  <  I  . 
Campani's  O.,    Airy's  O.  ;   Fr.   Oculaire   d'Huygens,  o.  de  Campani;  Ger.   Huy- 
gens'sches  Okular,   Cam]  aniches  Okular,  see  \  35.     Index  Ocular  ;  Ger.  Spitzen- 
O.     An  ocular  with  a  minute  pointer  or  two  pointers  at  the  level  of  the  real  image. 
The  points  are  movable  and  serve  for  indicators  and  also,  although  not  satisfacto- 
rily for  micrometry,     k'ellner's  Ocular,  see  orthoscopic  ocular.     Lore  Ocular,  also 
called  shallow  ocular.      An  ocular  which  magnifies  the  real  image  only  moderate- 
ly, i.e.,  2  to  S  fold.     Microm  !er  or  micrometric  Ocular;  Fr.  Oculaire  microme- 
trique   on  a   micrometre;    Ger.    Mikrometer-Okular,    Mess  Okular,    Bdneches   <>. 
Jackson  m.  o.,  see  \  38.     Micioscopic  Ocular  ;  Fr.  Oculaire  microscopic  ;  Ger.  mi- 
kroskopisches  Okular.     An  ocular  for  the  microscope  instead  of  one  for  a  telescope. 
Negative  Ocular,  see  i-  33.     Nelson's  s  :rew-micrometer  ocular.     A  modification  of 
the  Ramsden's  screw  or  cob-web  micrometer  in  which  positive  compensating  ocu- 
lars may  be  used.      Orthoscopic  Oculars;  also   called    Kelluer's   Ocular      1:    Ocu- 
laire orthoscopique  ;  Ger.  Kelluer'sches  oder  orthoskopisches  <>kular.     Au 
with  an  eye-lens  like  one  of  the  combinations  of  an  objective     I  and  a 

able  convex  field  lens.     The  field-lens  is  in  the  focus  of  tin-  eye  lens  and  thi 


24 


MICROSCOPE  AND  ACCESSORIES. 


\_CH.  I. 


\  35.  Huygenian  Ocular. — A  negative  ocular  designed  by  Huygens  for  the  tele- 
scope, but  adapted  also  to  the  microscope.  It  is  the  one  now  most  commonly  em- 
ployed. It  consists  of  a  field-lens  or  collective  (Fig.  30),  aiding  the  objective  in 
forming  the  real  image,  and  an  eye-lens  which  magnifies  the  real  image.     While 


Ocular  lo  2 


Fig.  31.  Compensating  Oculars  of  Zeiss,  with  section  removed  to  show  the  con- 
struction. The  litie  A- A  is  at  the  level  of  the  upper  end  of  the  tube  of  the  micro- 
scope zvhile  B-B  represents  the  lower  focal  points.  It  zvill  be  seen  that  the  mount- 
ing is  so  arranged  that  the  lower  focal  points  in  all  are  in  the  same  plane  and 
therefore  the  in  icroscope  remains  in  focus  upon  changing  octelars.  (  The  oculars  are 
par-focal).  The  lower  oculars,  2,  4  and  6  are  negative,  and  the  higher  ones,  8,  12 
iS,  are  positive.  The  numbers  2,  4.  6,  S,  12,  iS,  indicate  the  magnification  of  the 
ocular.     {From  Zeiss'  Catalog  No.  30). 


is  no  diaphragm  present.  The  field  is  large  and  flat.  Par-focal  Oculars,  a  series 
of  oculars  so  arranged  that  the  microscope  remains  in  focus  when  the  oculars  are 
interchanged  (Pennock,  Micr.  Bulletin,  vol.  iii,  p.  9,  31).  Periscopic  Ocular  ;  Fr. 
Oculaire  periscopique  ;  Ger.  periskopisches  Okular.  A  positive  ocular  devised  by 
Guudlach.  It  consists  of  a  double  convex  field  lens  and  a  triplet  eye-lens.  It 
gives  a  large,  flat  field.  Positive  Ocular,  see  \  34.  Projection  Ocular  ;  Fr.  Ocu- 
laire de  projection  ;  Ger.  Projections-Okular,  see  \  37.  Ramsden' s  Ocular  ;  Fr. 
Oculaire  de  Ramsden  ;  Ger.  Ramsden'sches  Okular.  A  positive  ocular  devised  by 
Ramsden.  It  consists  of  two  plano-convex  lenses  placed  close  together  with  the 
convex  surfaces  facing  each  other.  Only  the  central  part  of  the  field  is  clear. 
Searching  Ocular  ;  Fr.  Oculaire  d'orientatiou  ;  Ger.  Sucher-Okular,  see  \  36. 
Shalloiu  Ocular,  see  low  ocular.  Solid  Ocular,  holosteric  O.  ;  Fr.  Oculaire  holo- 
stere  ;  Ger.  holosterisches  Okular,  Vollglass-Okular.  A  negative  eye-piece  de- 
vised by  Tolles.  It  consists  of  a  solid  piece  of  glass  with  a  moderate  curvature  at 
one  end  for  a  field-lens,  and  the  other  end  with  a  much  greater  curvature  for  an  eye- 
lens.  For  a  diaphragm,  a  groove  is  cut  at  the  proper  level  and  filled  with  black 
pigment.  It  is  especially  excellent  where  a  high  ocular  is  desired.  Spectral  or 
spectroscopic  Ocular  ;  Fr.  Oculaire  spectroscopique  ;  Ger.  Spectral-Okular,  see  Mi- 
crospectroscope,  Ch.  VI.  Stauroscopic  Ocular  ;  Fr.  Oculaire  Stauroscopique. 
Ger.  Stauroskop-Okular.  An  ocular  with  a  Bertrand's  quartz  plate  for  mineralog- 
ical  purposes.  Working  Ocular;  Fr.  Oculaire  de  travail;  Ger.  Arbeits  Okular, 
see  I  36. 


PROPERTY  LIBRARY 

#,  C.  State  College 


CI  I.  /] 


Mli  'KOSCOPE  AND  ACCESSORIES. 


the  field  lens  aids  the  objective  in  the  formation  of  the  real,  inverted  image,  and 
increases  the  field  of  view,  it  also  combines  with  the  eye-lens  in  rendering  the  im- 
age achromatic. 

\  36.  Compensating  Oculars. — These  are  oculars  specially  constructed  for  use 
with  the  apochromatic  objectives.  They  compensate  for  aberrations  outside  tin- 
axis  which  could  not  be  so  readily  eliminated  in  the  objective  itself.  An  ocular 
of  tins  kind,  magnif  iug  but  twice,  is  made  for  use  with  high  powers,  for  the 
sake  of  the  large  field  in  finding  objects;  it  is  called  a  searching  ocular;  thi 
ordinarily  used  for  observation  are  in  contradistinction  called  working  ocula 
Part  of  the  compensating  oculars  are  positive  and  part  negative  (Fig.  31    . 

\  37.    Projection  Oculars. — These  are  oculars  especially  designed   for  projecting 
a  microscopic  image  on  the  screen  for  class  demonstrations,  or  for  photograph 
with  the  microscope.     While  they  are  specially  adapted  for  use  with  apochromatic 
objectives,  they   may  also   be  used  with  ordinary  achromatic  objectives  of  la 
numerical  aperture. 

Fig.  32.  Projection  Oculars  with  section  re- 
moved to  show  the  construction.  Below  are 
shown  the  upper  cuds  with  graduated  circle  to  in- 
dicate the  amount  of  rotation  found  necessary  to 
focus  the  diaphragm  on  the  screen.  Ao.  2,  No. 
/.  The  numbers  indicate  the  amount  the  ocular 
magnifies  the  image  formed  by  the  objective  as 
with  the  compensation  oculars.  |  Zeiss'  Catalog, 
No.  jo]. 

%  3S.  Micrometer  Ocular.— This  is  an  ocular 
connected  with  an  ocular  micrometer.  The 
micrometer  may  be  removable,  or  it  may  be  per- 
manently in  connection  with  the  ocular,  and  ar- 
ranged with  a  spring  and  screw,  by  winch  it  may 
be  moved  back  and  forth  across  the  field.  (See 
Ch.  IV). 


No.  2 


Fig.  33.  fig.  34. 

Figs.  33-34  Ocular  Micrometer  with  movable  scale.  Fig.  33  is  a  tide  \ 
the  ocular  while  Fig.  34.  gives  a  sectional  end  view,  and  shows  the  ocular  mi 
tei  in  position.     In  both  the  screw  which  moves  the  micrometer  i  tat  the 

left.     {From  Bausch  &  Lomb  Opt.  Co. ) 


26 


MICROSCOPE  AND  ACCESSORIES. 


\CH.  I. 


Fig.  35.  Ocular  Screw-Micrometer  7vith 
compensation  ocular  6.  The  tipper  figure 
shows  a  sectional  view  of  the  ocular  and  the 
screw  for  moving  the  micrometer  at  the  right. 
At  the  left  is  shown  a  clamping  screzv  to 
fasten  the  ocular  to  the  upper  part  of  the  mi- 
croscope tube.  Below  is  a  face  vieza,  showing 
the  graduation  on  the  wheel.  An  ocular 
micrometer  like  this  is  in  general  like  the 
cob- web  micrometer  and  may  be  used  for 
measuring  objects  of  varying  sizes  very  accu- 
rately. With  the  ordinary  ocular  microme- 
ter very  small  objects  frequently  fill  but  a  part 
of  an  interval  of  the  micrometer,  but  with  this 
the  movable  cross  lines  traverse  the  object  {or 
rather  its  realimage)  regardless  of  the  minute- 
ness of  the  object.     {Zeiss'  Catalog,  No.  30). 

§  39.   Spectral  or  Spectroscopic  Ocular. — (See  Micro-Spectroscope,  Ch.  VI). 

DESIGNATION    OF    OCULARS. 

1.  Equivalent  Focus. — As  with  objectives,  some  opticians  designate  the  ocu- 
lars by  their  equivalent  focus  (g  13).  With  this  method  the  power  of  the  ocular, 
as  with  objectives,  other  lenses  or  lens  systems,  varies  inversely  as  the  equivalent 
focal  length,  and  therefore  the  greater  the  equivalent  focal  length  the  less  the 
magnification.  This  seems  as  desirable  a  mode  for  oculars  as  for  objectives  and  is 
coming  more  and  more  into  use  by  the  most  progressive  opticians.  It  is  the 
method  of  designation  advocated  by  Dr.  R.  H.  Ward  for  many  years,  and  was 
recommended  by  the  committee  of  the  American  Microscopical  Society,  (Proc. 
Amer.  Micr.  Soc,  1SS3,  p.  175,  1SS4,  p.  22S). 

§41.  Numbering  and  Lettering. — Oculars  like  objectives  may  be  numbered  or 
lettered  arbitrarily.  When  so  designated,  the  smaller  the  number,  or  the  earlier 
the  letter  in  the  alphabet,  the  lower  the  power  of  the  ocular. 

\  42.  Magnification. — The  compensating  oculars  are  marked  with  the  amount 
the}'  magnify  the  real  image.  Thus  an  ocular  marked  X  4,  indicates  that  the  real 
image  of  the  objective  is  multiplied  four  fold  by  the  ocular. 

The  projection  oculars  are  designated  simply  by  the  amount  they  multiply  the 
real  image  of  the  objective.  Thus  for  the  short  or  160  nun.  tube-length  they  are, 
X  2,  X  4  ;  and  for  the  long  or  250  mm.  tube,  they  are  X  3  and  X  6.  That  is,  the 
final  image  on  the  screen  or  the  ground  glass  of  the  photographic  camera  will  be 
2,  3,  4,  or  6  times  greater  than  it  would  be  if  no  ocular  were  used.     See  Ch.  VIII. 

COMPOUND  MICROSCOPE. 

EXPERIMENTS. 


§43- 


Putting  an  Objective  in  Position  and  Removing  it. — Ele- 
vate the  tube  of  the  microscope  by  means  of  the  coarse  adjustment 
( frontispiece)  so  that  there  may  be  plenty  of  room  between  its  lower 


CH.  /.]  MICROSCOPE  AND  ACCESSORIES.  27 

end  and  the  stage.  Grasp  the  objective  lightly  near  its  lower  end  with 
two  fingers  of  the  left  hand,  and  hold  it  against  the  nut  in  the  lower 
end  of  the  tube.  With  two  fingers  of  the  right  hand  take  hold  of  the 
milled  ring  near  the  hack  or  upper  end  of  the  objective  and  screw  it 
into  the  tube  of  the  microscope.  Reverse  this  operation  for  removing 
the  objective.  By  following  this  method  the  danger  of  dropping  the 
objective  will  be  avoided. 

§  44.  Putting  an  Ocular  in  Position  and  Removing  it.  —  Elevate 
the  body  of  the  microscope  with  the  coarse  adjustment  so  that  the  ob- 
jective will  be  2  cm.  or  more  from  the  object — grasp  the  ocular  by  the 
milled  ring  next  the  eyedens  (Fig.  21),  and  the  coarse  adjustment  or 
the  tube  of  the  microscope  and  gently  force  the  ocular  into  position. 
In  removing  the  ocular,  reverse  the  operation.  If  the  above  precau- 
tions are  not  taken,  and  the  oculars  fit  snugly,  there  is  danger  in  insert- 
ing them  of  forcing  the  tube  of  the  microscope  downward  and  the  ob- 
jective upon  the  object. 

£  45.  Putting  an  Object  under  the  Microscope. — This  is  so  plac- 
ing an  object  under  the  simple  microscope,  or  on  the  stage  of  the  com- 
pound microscope,  that  it  will  be  in  the  field  of  view  when  the  micro- 
scope is  in  focus  (  ^  46). 

With  low  powers,  it  is  not  difficult  to  get  an  object  under  the  micro 
scope.  The  difficulty  increases,  however, 
with  the  power  of  the  microscope  and  the 
smallness  of  the  object.  It  is  usually  neces- 
sary to  move  the  object  in  various  directions 
while  looking  into  the  microscope,  in  order 
to  get  it  into  the  field.  Time  is  usually 
saved  by  getting  the  object  in  the  center  of 
the  field  with  a  low  objective  before  putting        fxar 

the    high    objective    in    position.       This    is       FlG-   3*.    Triple  nose-piece 

.,     r     .,.,    ,ii-                         •  or  revolver  for  quickly  chang- 

greatly  meditated  bv  using  a  nose-piece,  or  .        ,  .    ..       , ',           .      . 

,                               ,  ing  objectives  {Queen  &  Co.). 
revolver.     (  See  also  $  118,  Figs.  61-66  1. 

§  46.  Field  or  Field  of  View  of  a  Microscope. — This  is  the  area 
visible  through  a  microscope  when  it  is  in  focus.  When  properly  lighted, 
and  there  is  no  object  under  the  microscope,  the  field  appears  as  a  circle 
of  light.  When  examining  an  object  it  appears  within  the  light  circle, 
and  by  moving  the  object,  if  it  is  of  sufficient  size,  different  parts  are 
brought  successively  into  the  field  of  view. 

In  general,  the  greater  the  magnification  of  the  entire  microscope, 
whether  the  magnification  is  produced  mainly  by  the  objective,  the 
ocular,  or  by  increasing  the  tube  length,  or  by  a  combination  of  all 
three  (see  Ch.  IV,  under  magnification  i,  the  smaller  is  the  field. 


28 


MICROSCOPE  AND  ACCESSORIES. 


\CH.  I 


The  size  of  the  field  is  also  dependent,  in  part,  without  regard  to 
magnification,  upon  the  size  of  the  opening  in  the  ocular  diaphragm. 
Some  oculars,  as  the  orthoscopic  and  periscopic,  are  so  constructed  as 
to  eliminate  the  ocular  diaphragm,  and  in  consequence,  although  this 
is  not  the  sole  cause,  the  field  is  considerably  increased.  The  exact 
size  of  the  field  may  be  read  off  directly  by  putting  a  stage  micrometer 
under  the  microscope  and  noting  the  number  of  spaces  required  to 
measure  the  diameter  of  the  light  circle. 

>j  47.  Table  shon  'ing  the  actual  size  in  millimeters  of  the  field  of  a  group 
of  commonly  used  objectives  aud  oculars.  Compare  with  the  graphic  rep- 
resentation in  Fig.  j~j.     See  also  §  /<5. 


Equivalent 

Focus  and 

N.  A.  of 

Objective. 

Diameter 

of  Field 

in  mm. 

Ocular. 

S5  mm.  .  .    . 

15-4 
10  6 

3.3 

llVz 

25 

12^ 

mm. 

1  i 

Huygeniau. 

45  mm.  .  .    . 

7.0 

50 
4.0 

37^ 
25 

I2>^ 

mm. 

Huygenian. 

17  mm.  .  .    . 
N.  A.  =0.25 

30 
2  0 
1.6 

25 
12^ 

mm. 

Huygeniau. 

5-7 
28 
1.4 
0.97 

180 

45 
15 
10 

mm. 
if 

Compensation. 

5  mm.  .    .    . 

N.A.  =0  92 

0541 
0371 
0.290 

37^ 
25 

I2j^ 

mm. 

1 1 

Huygenian. 

0S50 
0501 
0  250 
0.173 

I  So 
45 
15 
10 

mm. 

1  t 

" 

Compensation. 

2  mm.  .    .    . 

N.A.  =  1.25 

0  270 

0.186 
0.147 

37^ 

25 

12^ 

u 

i  « 
x 

Huygeniau. 

0.450 
0.251 
0.125 
0.088 

180 
45 
15 
10 

mm. 

4 1 

1 1 
i  1 

Compensation. 

CH.  /.]  MICROSCOPE  AND  ACCESSORIES. 


2    fft  m 
\ 


45  mm  '?  mm 

8  j    m  m 


S   "i  m 


Fig.  37.  Figures  showing  approximately  the  actual  size  of  the  field  with  ob- 
jectives of 85  mm.,  45  mm.,  ij  mm.,  5  mm.,  and  _■  mm.,  equivalent  focus,  and 

ocular  of  3jYz  mm.,  equivalent  focus  in  each  case.      This  figure  shows  graphically 
what  is  also  very  clearly  indicated  in  the  table  {\  47). 

§  48.  The  size  of  the  field  of  the  microscope  as  projected  into  the 
field  of  vision  of  the  normal  human  eye  (/.  c. ,  the  virtual  image  1  may 
be  determined  by  the  use  of  the  camera  lucida  with  the  drawing  surface 
placed  at  the  standard  distance  of  250  millimeters  (Ch.  IV). 

FUNCTION    OF    AN    OBJECTIVE. 

^  49.  Put  a  2-in.  (50  mm.)  objective  on  the  microscope  or  screw  off 
the  front  combination  of  a  16  mm.,  (  23-in.  ),  and  put  the  back-combina- 
tion on  the  microscope  for  a  low  objective. 

Place  some  printed  letters  or  figures  under  the  microscope,  and  light 
well.  In  place  of  an  ocular,  put  a  screen  of  ground  glass,  or  a  piece  of 
lens  paper,  over  the  upper  end  of  the  tube  of  the  microscope.* 

Lower  the  tube  of  the  microscope  by  means  of  the  coarse  adjustment 
until  the  objective  is  within  2-3  cm.  of  the  object  on  the  stage.  Look 
at  the  screen  on  the  top  of  the  tube,  holding  the  head  about  as  far  from 
it  as  for  ordinary  reading,  and  slowly  elevate  the  tube  by  means  of  the 
coarse  adjustment  until  the  image  of  the  letter  appears  on  the  screen. 

The  image  can  be  more  clearly  seen  if  the  object  is  in  a  strong  light 
and  the  screen  in  a  moderate  light,  i.  c,  if  the  top  of  the  microscope  is 
shaded. 

The  letters  will  appear  as  if  printed  on  the  ground  glass  or  paper,  but 
will  be  inverted  (Fig.  21). 

If  the  objective  is  not  raised  sufficiently,  and  the  head  is  held  too 
near  the  microscope,  the  objective  will  act  as  a  simple  microscope.  If 
the  letters  are  erect,  and  appear  to  be  down  in  the  microscope  and  not 
on  the  screen,  hold  the  head  farther  from  it,  shade  the  screen,  and  raise 
the  tube  of  the  microscope  until  the  letters  do  appear  on   the  ground 


glass. 


*  Ground  glass  may  be  very  easily  prepared  by  placing  some  fine  emery  between 
two  pieces  of  glass,  wetting  it  with   water  ami  then   rubbing   the  glasi  .ether 

for  a  few  minutes.     If  the  glass  becomes  too  opaque,  it  may   be  rendered  more 
trauslucent  by  rubbing  some  oil  upon  it. 


30  MICROSCOPE  AND  ACCESSORIES.  [CII.  I. 

To  demonstrate  that  the  object  must  be  outside  the  principal  focus 
with  the  compound  microscope,  remove  the  screen  and  turn  the  tube  of 
the  microscope  directly  toward  the  sun.  Move  the  tube  of  the  micro- 
scope with  the  coarse  adjustment  until  the  burning  or  focal  point  is  found 
I  $  6).  Measure  the  distance  from  the  paper  object  on  the  stage  to  the 
objective,  and  it  will  represent  approximately  the  principal  focal  dis- 
tance (Figs.  10,  n).  Replace  the  screen  over  the  top  of  the  tube,  no 
image  can  be  seen.  Slowly  raise  the  tube  of  the  microscope  and  the 
image  will  finally  appear.  If  the  distance  between  the  object  and  the 
objective  is  now  taken,  it  will  be  found  considerably  greater  than  the 
principal  focal  distance  (compare  §  9). 

£  50.  Aerial  Image. — After  seeing  the  real  image  on  the  ground- 
glass,  or  paper,  use  the  lens  paper  over  about  half  of  the  opening  of 
the  tube  of  the  microscope.  Hold  the  eye  about  250  mm.  from  the 
microscope  as  before  and  shade  the  top  of  the  tube  by  holding  the  hand 
between  it  and  the  light,  or  in  some  other  way.  The  real  image  can 
be  seen  in  part  as  if  on  the  paper  and  in  part  in  the  air.  Move  the 
paper  so  that  the  image  of  half  a  letter  will  be  on  the  paper  and  half 
in  the  air.  Another  striking  experiment  is  to  have  a  small  hole  in  the 
paper  placed  over  the  center  of  the  tube  opening,  then  if  a  printed  word 
extends  entirely  across  the  diameter  of  the  tube  its  central  part  may  be 
seen  in  the  air,  the  lateral  parts  on  the  paper.  The  advantage  of  the 
paper  over  part  of  the  opening  is  to  enable  one  to  accommodate  the 
eyes  for  the  right  distance.  If  the  paper  is  absent  the  eyes  adjust 
themselves  for  the  light  circle  at  the  back  of  the  objective,  and  the 
aerial  image  appears  low  in  the  tube.  Furthermore,  it  is  more  difficult 
to  see  the  aerial  image  in  space  than  to  see  the  image  on  the  ground- 
glass  or  paper,  for  the  eye  must  be  held  in  the  right  position  to  receive 
the  rays  projected  from  the  real  image,  while  the  granular  surface  of 
the  glass  and  the  delicate  fibers  of  the  paper  reflect  the  rays  irregularly, 
so  that  the  image  may  be  seen  at  almost  any  angle,  as  if  the  letters 
were  actually  printed  on  the  paper  or  glass. 

§51.  The  function  of  an  objective,  as  seen  from  these  experi- 
ments, is  to  form  an  enlarged,  inverted,  real  image  of  an  object,  this 
image  being  formed  on  the  opposite  side  of  the  objective  from  the  ob- 
ject (Fig.  21). 

FUNCTION    OF    AN    OCULAR. 

§  52.  Using  the  same  objective  as  for  §  49,  get  as  clear  an  image  of 
the  letters  as  possible  on  the  lens  paper  screen.  Look  at  the  image 
with  a  simple  microscope  (Fig.  17  or  18)  as  if  the  image  were  an  object. 


CI  I.  /.] 


MICROSCOPE  AND  ACCESSORIES. 


Observe  that  the  image  seen  through  the  simple  microscope  is  merely 
an  enlargement  of  the  one  <>n  the  screen,  and  that  the  letters  remain 
inverted,  that  is  they  appear  as  with  the  naked  eye  *  §  9  1.  Remove  the 
screen  and  observe  the  aerial  image  with  the  tripod. 

Put  a  50  111111.,  (A,  No.  1,  or  2  in.)  ocular  < /.  <■.,  an  ocular  of  low 
magnification)  in  position  (§  44).  Hold  the  eye  about  10  to  20  milli- 
meters from  the  eye-lens  and  look  into  the  microscope.  The  letfc 
will  appear  as  when  the  simple  microscope  was  used  1  see  above  ),  the 
image  will  become  more  distinct  by  slightly  raising  the  tube  of  the  mi- 
croscope with  the  coarse  adjustment. 

§  53.  The  Function  of  the  Ocular,  as  seen  from  the  above,  is  that 
of  a  simple  microscope,  viz.  :  It  magnifies  the  real  image  formed  by  the 
objective  as  if  that  image  were  an  object.  Compare  the  image  formed 
by  the  octdar  (Fig.  21),  and  that  formed  by  a  simple  microscope  (Fig. 

38). 

It  should  be  borne  in  mind,  however,  that  the  rays  from  an  object  as 
usually  examined  with  a  simple  microscope,  extend  from  the  object  in 
all  directions,  and  no  matter  at  what  angle  the  simple  microscope  is  held, 
provided  it  is  sufficiently  near  and  points  toward  the  object,  an  image 
may  be  seen.     The  rays  from  a  real  image,  however,   are  continued  in 

Fig.  38.  Diagram  of  the  simple  microscope  show 
ing  the  course  of  the  rays  and  all  the  images,  and 
that  the  eye  forms  an  integral  part  of  it. 

A1  />".  The  object  within  the  principal  focus.  As 
/!'■.  The  virtual  image  on  the  same  side  of  the  lens 
as  the  object.  It  is  indicated  with  dotted  lines,  as 
it  has  no  actual  existence. 

B-  A1.  Retinal  image  of  the  object  (A1  Bl ).  Tin- 
virtual  image  is  simply  a  projection  of  the  retinal 
image  in  the  field  of  vision. 

Axis.  The  principal  optic  axis  of  the  microscope 
and  of  the  eye.  Cr.  Cornea  of  the  eye.  L.  Crystal- 
line lens  of  the  eye.  R.  Ideal  refracting  surface  at 
which  all  the  refractions  of  the  eye  may  he  assumed 
to  take  place. 

certain  definite  lines  and  not  in  all  directions  ; 

hence,  in  order  to  see  this  aerial  image  with 

an  ocular  or  simple  microscope,  or  in  order 

to  see  the  aerial  image  with  the  unaided  eye,  the  simple  microscope, 

ocular  or  eye  must  be  put  in  the  path  of  the  rays  (  Fig.  2  1 

S  54.   The   field-lens  of  a   Iluygvnian  ocular  makes  the  real  image 
smaller  and  consequently  increases  the  size  of  the  field  ;  it  also  ma',. 
the  image  brighter  by  contracting  the  area  of  the  real  image     I 


32  MICROSCOPE  AND  ACCESSORIES.  [CH.  I. 

Demonstrate  this  by  screwing  off  the  field-lens  and  using  the  eye-lens 
alone  as  in  the  ocular,  refocusing  if  necessary.  Note  also  that  the  let- 
ters or  other  image  is  bordered  by  a  colored  haze  (§7). 

When  looking  into  the  ocular  with  the  field-lens  removed,  the  eye 
should  not  be  held  so  close  to  the  ocular,  as  the  eye-point  is  consider- 
ably farther  away  than  when  the  field-lens  is  in  place. 

^  55.  The  eye-point. — This  is  the  point  above  the  ocular  or  simple 
microscope  where  the  greatest  number  of  emerging  rays  cross.  Seen 
in  profile,  it  may  be  likened  to  the  narrowest  part  of  an  hour  glass. 
Seen  in  section  (Fig.  30),  it  is  the  smallest  and  brightest  light  circle 
above  the  ocular.  This  is  called  the  eye-point,  for  if  the  pupil  of  the 
eve  is  placed  at  this  level,  it  will  receive  the  greatest  number  of  rays 
from  the  microscope,  and  consequently  see  the  largest  field. 

Demonstrate  the  eye-point  by  having  in  position  an  objective  and 
ocular  as  above  (§  49).  Light  the  object  brightly,  focus  the  micro- 
scope, shade  the  ocular,  then  hold  some  ground-glass  or  a  piece  of  the 
lens  paper  above  the  ocular  and  slowly  raise  and  lower  it  until  the 
smallest  circle  of  light  is  found.  By  using  different  oculars  it  will  be 
seen  that  the  eye-point  is  nearer  the  eye-lens  in  high  than  in  low  ocu- 
lars, that  is  the  eye-point  is  nearer  the  eye-lens  for  an  ocular  of  small 
equivalent  focus  than  for  one  of  greater  focal  length. 

REFERENCES    FOR   CHAPTER   I. 

In  the  appendix  will  be  given  a  bibliography,  with  full  titles,  of  the  works  and 
periodicals  referred  to. 

For  the  subjects  considered  in  this  chapter  general  works  on  the  microscope 
may  be  consulted  with  great  advantage  for  different  or  more  exhaustive  treatment. 
The  most  satisfactory  work  in  English  is  Carpenter-Dallinger.  For  the  history  of 
the  microscope,  Mayall's  Cantor  Lectures  on  the  microscope  are  very  satisfactorv. 
See  also  Bjale,  E  Bausch,  Beherens,  Kossel  and  Schiefferdecker,  Dippel,  Frev, 
Harting,  Hogg,  Nageli  and  Schweudener,  Robin,  Van  Heurck,  Clark,  Cross  and 
Cole,  vStokes. 

The  following  special  articles  in  periodicals  may  be  examined  with  advantage  : 

Apochromatic  Objectives,  etc.  Dippel  in  Zeit.  wiss.  Mikr.,  1SS6,  p.  303  ;  also  in 
the  Jour.  Roy.  Micr.  Soc,  1886,  pp.  316,  S49,  1110  ;  same,  1S90,  p.  4S0  ;  Zeit.  f.  In- 
strumentenk.,  1890,  pp.  1-6;  Micr.  Built.,  1891,  pp.  6-7. 

Tube-length,  etc.  Gage,  Proc.  Amer.  Soc.  Micrs.,  1887,  pp.  168-172  ;  also  in  the 
Microscope,  the  Jour.  Roy.  Micr.  Soc,  and  in  Zeit.  wiss.  Mikr.,  1887-8,  Bausch, 
Proc.  Amer.  Soc.  Micrs.,  1890,  pp.  43-49  ;  also  in  the  Microscope,  1890,  pp.  2S9-296. 

Aperture.  J.  D.  Cox,  Presidential  Address,  Proc.  Amer.  Soc.  Micrs.,  1884,  pp. 
5-39.  Jour.  Roy.  Micr.  Soc,  18S1,  pp.  303,  348,  365,  388  ;  1882,  pp.  300,  460  ;  1S83, 
p.  790  ;   1884,  p.  20. 


CHAPTER  II. 


LIGHTING    AND    FOCUSING;     MANIPULATION    OF    DRY, 

ADJUSTABLE    AND    IMMERSION    OBJECTIVES;    CAR  I. 

OF    THE    MICROSCOPE    AND    OF    THE    EYES; 

LABORATORY    MICROSCOPES. 


APPARATUS   AND   MATERIAL   FOR   THIS   CHAPTER. 

Microscope  supplied  with  plane  and  concave  mirror,  achromatic  and  Abbe  con- 
densers, dry,  adjustable  and  immersion  objectives,  oculars,  tripple  nose-piece. 
Microscope  lamp  and  movable  condenser  (bull's  eye  or  other  form  (Fig.  52), 
Homogeneous  immersion  liquid;  Benzin,  alcohol,  distilled  water;  Mounted 
preparation  of  fly's  wing  ($  68)  ;  Mounted  preparation  of  Pleurasigma.  Stage  or 
ocular  micrometer  with  lines  filled  with  graphite  {\  73,  74)  ;  Glass  slides  and 
cover-glasses  (Ch.  VII)  ;  10  per  ct.  solution  of  salicylic  acid  in  95  per  ct.  alcohol 
{\  88)  ;  Preparation  of  stained  microbes  ($  101)  ;  Vial  of  equal  parts  olive  or  cot- 
ton seed  oil  or  liquid  vaselin  and  benzin  ($  105)  ;  Ward's  and  double  eye-shade 
(Figs.  59,  60)  ;  Screen  for  whole  microscope  (Fig.  58). 

FOCUSING. 

\  56.  Focusing  is  mutually  arranging  an  object  and  the  microscope  so  that  a 
clear  image  may  be  seen. 

With  a  simple  microscope  (?  9)  either  the  object  or  the  microscope  or  both  may 
be  moved  in  order  to  see  the  image  clearly,  but  with  the  compound  microscope 
the  object  more  conveniently  remains  stationary  on  the  stage,  and  the  tube  or 
body  of  the  microscope  is  raised  or  lowered  (frontispiece). 

In  general,  the  higher  the  power  of  the  whole  microscope  whether  simple  or 
compound,  the  nearer  together  must  the  object  and  objective  be  brought.  With 
the  compound  microscope,  the  higher  the  objective,  and  the  longer  the  tube  of 
the  microscope,  the  nearer  together  must  the  object  and  the  objective  be  brought. 
If  the  oculars  are  not  par-focal,  the  higher  the  magnification  of  the  ocular,  the 
nearer  must  object  and  objective  be  brought. 

\  57-  Working  Distance.  —  By  this  is  meant  the  space  between  the  simple  micro- 
scope and  the  object,  or  between  the  front  lens  of  the  compound  microscope  and 
the  object,  when  the  microscope  is  in  focus.  This  working  distance  is  always  con- 
siderably less  than  the  equivalent  focal  length  of  the  objective.  For  example, 
the  front-lens  of  a  %\\\  in.,  or  6  mm.  objective  would  not  be  '4  th  inch,  or  <i  milli- 
meters from  the  object  when  the  microscope  is  in  focus,  but  considerably  less  than 
that  distance.     If  there  were  no  other  reason  than  the  limited  working  distance  of 

3 


34 


LIGHTING  AND  FOCUSING. 


\_CH.  II. 


high  objectives,  it  would  be  necessary  to  use  ver}'  thin  cover-glasses  over  the  ob- 
ject. (See  \  22,  27).  If  too  thick  covers  are  used,  it  may  be  impossible  to  get  an 
objective  near  enough  an  object  to  get  it  in  focus.  For  objects  that  admit  of  ex- 
amination with  high  powers  it  is  always  better  to  use  thin  covers. 

LIGHTING   WITH   DAYLIGHT. 

\  58.  Unmodified  sunlight  should  not  be  employed  except  in  special  cases. 
North  light  is  best  and  most  uniform.  When  the  sky  is  covered  with  white  clouds 
the  light  is  most  favorable.  To  avoid  the  shadows  produced  by  the  hands  in 
manipulating  the  mirror,  etc.,  it  is  better  to  face  the  light  ;  but  to  protect  the  eyes 
and  to  shade  the  stage  of  the  microscope  some  kind  of  screen  should  be  used. 
The  one  figured  in  (Fig.  58)  is  cheap  and  efficient.  If  one  dislikes  to  face  the 
window  or  lamp  it  is  better  to  sit  so  that  the  light  will  come  from  the  left  as  in 
reading. 

It  is  of  the  greatest  importance  and  advantage  for  one  who  is  to  use  the  micro- 
scope for  serious  work  that  he  should  comprehend  and  appreciate  thoroughly  the 
various  methods  of  illumination,  and  the  special  appearances  due  to  different 
kinds  of  illumination. 

Depending  on  whether  the  light  illuminating  an  object  traverses  the  object  or  is 
reflected  upon  it,  and  also  whether  the  object  is  symmetrically  lighted,  or  lighted 
more  on  one  side  than  the  other,  light  used  in  microscopy  is  designated  as  re- 
flected and  transmitted,  axial  and  oblique. 


rt3.g 


39-  40. 

Figs.  39-40.  For  full  explanation  see  Figs.  22  and  23. 

\  59.  Reflected,  Incident  or  Direct  Light.— By  this  is  meant  light  reflected  upon 
the  object  in  some  way  and  then  irregularly  reflected  from  the  object  to  the  micro- 
scope.    By  this  kind  of  light  objects  are  ordinarily  seen  by  the  unaided  eye,  and 


CI  I.  II.  ]  /.  IGHTING  AND  FOL  l  SIXG.  35 

the  objects  are  mostly  opaque.     In  Vertebrate  Histology,  reflected  light   is  but 

little  used  ;  but  in  the  study  of  opaque  objects,  like  whi  1  ts,  etc.,  it  is  used 

a  great  deal.  For  low  powers,  ordinary  daylight  that  naturally  falls  upon  the  ob- 
ject, or  is  reflected  or  condensed  upon  it  with  a  mirror  <>r  condensing  lens,  an  sue:-- 
very  well.  For  high  powers  and  for  special  purposes,  special  illuminating  appa- 
ratus has  been  devised  (2  26).     (See  also  Carpenter- Dallin^cr,  p.  278). 

0.  Transmitted  Light.  —  By  this  is  meant  light  which  passes  through  an  ob- 
ject from  the  opposite  side.  The  details  of  a  photographic  negative  are  in  many 
cases  only  seen  or  best  seen  by  transmitted  light,  while  the  print  made  from  it  is 
best  seen  by  reflected  light. 

Almost  all  objects  studied  in  Vertebrate  Histology  are  lighted  by  transmitted 
light,  and  they  are  in  some  way  rendered  transparent  or  semi  transparent.  The 
light  traversing  and  serving  to  illuminate  the  object  in  working  with  a  compound 
microscope  is  usually  reflected  from  a  plane  or  concave  mirror,  or  fiom  a  mirror  to 
a  condenser  (?  75),  and  thence  transmitted  to  the  object  from  below  (Figs.  4S-51). 

\  61.  Axial  or  Central  Light.  —  By  this  is  understood  light  reaching  the  object, 
the  rays  of  light  being  parallel  to  each  other  and  to  the  optic  axis  of  the  micro- 
scope, or  a  diverging  or  converging  cone  of  light  whose  axial  ray  is  parallel  with 
the  optic  axis  of  the  microscope.  In  either  case  the  object  is  symmetrically  illu- 
minated. 

\  62.  Oblique  Light. — This  is  light  in  which  parallel  rays  from  a  plane  mirror 
form  an  angle  with  the  optic  axis  of  the  microscope  (Fig.  40).  Or  if  a  concave 
mirror  or  a  condenser  is  used,  the  light  is  oblique  when  the  axial  ray  of  the  cone 
of  light  forms  an  angle  with  the  optic  axis  (Fig.  49). 

DIAPHRAGMS. 

\  63.  Diaphragms  and  their  Proper  Employment. — Diaphragms  are  opaque  disks 
with  openings  of  various  sizes,  which  are  placed  between  the  source  of  light  or 
mirror  and  the  object.  In  some  cases  an  iris  diaphragm  is  used,  and  then  the  same 
one  is  capable  of  giving  a  large  range  of  openings.  The  object  of  a  diaphragm,  in 
general,  is  to  cut  off  all  adventitious  light  and  thus  enable  one  to  light  the  object 
in  such  a  way  that  the  light  finally  reaching  the  microscope  shall  all  come  from 
the  object  or  its  immediate  vicinity.  The  diaphragms  of  a  condenser  serve  to  vary 
its  aperture  to  the  needs  of  each  object  and  each  objective. 

\  64.  Size  and  Position  of  Diaphragm  Opening. — When  no  condenser  is  used 
the  size  of  the  opening  in  the  diaphragm  should  be  about  that  of  the  front  lens  of 
the  objective  used.  For  some  objects  and  some  objectives  this  rule  may  be  quite 
widely  departed  from  ;  one  must  learn  by  trial. 

When  lighting  with  a  mirror  the  diaphragm  should  be  as  close  as  possible  to  the 
object  in  order,  (a)  that  it  may  exclude  all  adventitious  light  from  the  object  ;  (b) 
that  it  may  not  interfere  with  the  most  efficient  illumination  from  the  mirror  by 
cutting  off  a  part  of  the  illuminating  pencil.  If  the  diaphragm  is  a  considerable 
distance  below  the  object,  (1)  it  allows  considerable  adventitious  light  to  reach  the 
object  and  thus  injures  the  distinctness  of  the  microscopic  image  ;  1  i;  prevents 
the  use  of  very  oblique  light  unless  it  swings  with  the  mirror  ;  ;  it  cuts  off  1  part 
of  the  illuminating  cone  from  a  concave  mil  tor.  On  the  Other  hand,  even  with  a 
small  diaphragm,  the  whole  field  will  be  lighted. 

With  an  illuminator  or  condenser  (Figs.  41,  4S  t,  the  diaphragm  serves  to  narrow 


36  LIGHTING  AND  FOCUSING.  [CH.  II. 

the  pencil  to  be  transmitted  through  the  condenser,  and  thus  to  limit  the  aperture 
or  for  any  special  purpose  to  be  served  (see  \  80).  Furthermore,  by  making  the 
diaphragm  opening  eccentric,  oblique  light  may  be  used,  or  by  using  a  diaphragm 
with  a  slit  around  the  edge  (central  stop  diaphragm),  the  center  remaining  opaque, 
the  object  may  be  lighted  with  a  hollow  cone  of  light,  all  of  the  rays  having  great 
obliquity.  In  this  way  the  so  called  dark-ground  illumination  may  be  produced 
(§88;  Fig.  51). 

ARTIFICIAL   ILLUMINATION. 

§  65.  For  evening  work  and  for  certain  special  purposes,  artificial  illumination 
is  employed.  A  good  petroleum  (kerosene)  lamp  with  fiat  wick  has  been  found 
very  satisfactory,  but  for  brilliancy  and  for  the  actinic  power  necessary  for  photo- 
micrography (see  Ch.  VIII)  the  new  acetylene  light  seems  to  be  all  that  could  be 
desired.  Whatever  source  of  artificial  light  is  employed,  the  light  should  be  bril- 
liant and  steady. 

LIGHTING   AND    FOCUSING  :    EXPERIMENTS. 

§  66.  Lighting  with  a  Mirror. — Place  a  mounted  fly's  wing  under 
the  microscope,  put  the  16  mm.  {2/i  in.)  or  other  low  objective  in  posi- 
tion, also  a  low  ocular.  With  the  coarse  adjustment,  lower  the  tube  of 
the  microscope  to  within  about  1  cm.  of  the  object.  Use  an  opening  in 
the  diaphragm  about  as  large  as  the  front  lens  of  the  objective  ;  then 
with  the  plane  mirror  try  to  reflect  light  up  through  the  diaphragm 
upon  the  object.  One  can  tell  when  the  field  (§  46)  is  illuminated,  by 
looking  at  the  object  on  the  stage,  but  more  satisfactorily  by  looking 
into  the  microscope.  It  sometimes  requires  considerable  manipulation 
to  light  the  field  well.  After  using  the  plane  side  of  the  mirror  turn 
the  concave  side  into  position  and  light  the  field  with  it.  As  the  con- 
cave mirror  condenses  the  light,  the  field  will  look  brighter  with  it  than 
with  the  plane  mirror.  It  is  especially  desirable  to  remember  that  the 
excellence  of  lighting  depends  in  part  on  the  position  of  the  diaphragm 
(§  57)-  If  the  greatest  illumination  is  to  be  obtained  from  the  concave 
mirror,  its  position  must  be  such  that  its  focus  will  be  at  the  level  of  the 
object.  This  distance  can  be  very  easily  determined  by  finding  the 
focal  point  of  the  mirror  in  full  sunlight. 

§  67.  Use  of  the  Plane  and  of  the  Concave  Mirror. — The  mirror 
should  be  freely  movable,  and  have  a  plane  and  a  concave  face.  The 
concave  face  is  used  when  a  large  amount  of  light  is  needed,  the  plane 
face  when  a  moderate  amount  is  needed  or  when  it  is  necessarjr  to  have 
parallel  rays  or  to  know  the  direction  of  the  rays. 

§  6S.  Focusing  with  Low  Objectives.  —  Place  a  mounted  fly's 
wing  under  the  microscope  ;  put  the  16  mm.  (2j  in.)  objective  in  posi- 
tion, and  also  the  lowest  ocular.  Select  the  proper  opening  in  the  dia- 
phragm and  light  the  object  well  with  transmitted  light  (§  60,  64). 


CH.  II.]  LIGHTING  AND  FOCUSING.  37 

Hold  the  head  at  about  the  level  of  the  stage,  look  toward  the  win- 
dow, and  between  the  object  and  the  front  of  the  objective- ;  with  the 
coarse  adjustment  lower  the  tube  until  the  objective  is  within  about 
half  a  cm.  of  the  object.  Then  look  into  the  microscope  and  slowly 
elevate  the  tube  with  the  coarse  adjustment.  The  image  will  appear 
dimly  at  first,  but  will  become  very  distinct  by  turning  the  tube  ^t ill 
higher.  If  the  tube  is  raised  too  high  the  image  will  become  indistinct, 
and  finally  disappear.  It  will  again  appear  if  the  tube  is  lowered  the 
proper  distance. 

When  the  microscope  is  well  focused  try  both  the  concave  and  the 
plane  mirrors,  in  various  positions  and  note  the  effect.  Put  a  high  ocu- 
lar in  place  of  the  low  one  (§  40).  If  the  oculars  are  not  par- focal  it 
will  be  necessary  to  lower  the  tube  somewhat  to  get  the  image  in  focus. * 

Pull  out  the  draw-tube  4-6  cm.,  thus  lengthening  the  body  of  the 
microscope,  and  it  will  be  found  necessary  to  lower  the  tube  of  the  mi- 
croscope somewhat.      (For  reason,  see  Fig.  57). 

§69.  Pushing  in  the  Draw-Tube. — To  push  in  the  draw-tube, 
grasp  the  large  milled  ring  of  the  ocular  with  one  hand,  and  the  milled 
head  of  the  coarse  adjustment  with  the  other,  and  gradually  push  the 
draw-tube  into  the  tube.  If  this  were  done  without  these  precautions 
the  objective  might  be  forced  against  the  object  and  the  ocular  thrown 
out  by  the  compressed  air. 

§70.  Focusing  with  High  Objectives.  —  Employ  the  same  object 
as  before,  elevate  the  tube  of  the  microscope  and  remove  the  16  mm. 
(^<3  in.)  objective  as  indicated.  Put  the  3  mm.  (  }i  in.)  or  a  higher  ob- 
jective in  place,  and  use  a  low  ocular. 

Light  well,  and  employ  the  proper  opening  in  the  diaphragm,  etc. 
(§  64).  Look  between  the  front  of  the  objective  and  the  object  as  be- 
fore (§  68),  and  lower  the  tube  with  the  coarse  adjustment  till  the  ob- 
jective almost  touches  the  cover-glass  over  the  object.  Look  into  the 
microscope,  and  with  the  coarse  adjustment,  raise  the  tube  very  slowly 
until  the  image  begins  to  appear,  then  turn  the  milled  head  of  the  fine 
adjustment  (frontispiece),  first  one  way  and  then  the  other,  if  neces- 
sary, until  the  image  is  sharply  defined. 

In  practice  it  is  found  of  great  advantage  to  move  the  preparal 
slightly  while  focusing.     This  enables  one  to  determine  the  approach 

*  Par-focal  oculars  are  so  constructed,  or  so  mounted,  that  those  of  diffirent  pow- 
ers may  he  interchanged  without  the  microscopic  image  becoming  wholly  out  of 
focus  (Fig.  31,  note,  p.  23).  When  high  objectives  are  used,  while  the  image  m.iy 
be  seen  after  changing  oculars,  the  instrument  nearly  always  needs  slight  focusing. 
With  low  powers  this  may  not  be  necessary. 


38  LIGHTING  AND  FOCUSING.  \CH.  II. 

to  the  focal  point  either  from  the  shadow  or  the  color,  if  the  object  is 
colored.  With  high  powers  and  scattered  objects  there  might  be  no  ob- 
ject in  the  small  field  (see  §  46,  Fig.  37,  for  size  of  field).  By  moving 
the  preparation  an  object  will  be  moved  across  the  field  and  its  shadow 
gives  one  the  hint  that  the  objective  is  approaching  the  focal  point.  It 
is  sometimes  desirable  to  focus  on  the  edge  of  the  cement  ring  or  on  the 
little  ring  made  by  the  marker  (see  Figs.  61-65  §  118). 

Note  that  this  high  objective  must  be  brought  nearer  the  object  than 
the  low  one,  and  that  by  changing  to  a  higher  ocular  (if  the  oculars  are 
not  par-focal)  or  lengthening  the  tube  of  the  microscope  it  will  be  found 
necesssary  to  bring  the  objective  still  nearer  the  object,  as  with  the  low 
objective.      (For  reason  see  Fig.  57). 

§  71.  Always  Focus  Up,  as  directed  above.  If  one  lowers  the  tube 
only  when  looking  at  the  end  of  the  objective  as  directed  above,  there 
will  be  no  danger  of  bringing  the  objective  in  contact  with  the  object, 
as  may  be  done  if  one  looks  into  the  microscope  and  focuses  down. 

When  the  instrument  is  well  focused,  move  the  object  around  in  order 
to  bring  different  parts  into  the  field  of  view  (§  46).  It  may  be  neces- 
sary to  re-focus  with  the  fine  adjustment  every  time  a  different  part  is 
brought  into  the  field.  In  practical  work,  one  hand  is  kept  on  the  fine 
adjustment  constantly,  and  the  focus  is  continually  varied. 

§  72.  Determination  of  Working  Distance. — As  stated  in  §  57 
this  is  the  distance  between  the  front  lens  of  the  objective  and  the  object 
when  the  objective  is  in  focus.  It  is  always  less  than  the  equivalent 
focal  length  of  the  objective. 

Make  a  wooden  wedge  10  cm.  long  which  shall  be  exceedingly  thin 
at  one  end  and  about  20  mm.  thick  at  the  other.  Place  a  slide  on  the 
stage  and  some  dust  on  the  slide.  Do  not  use  a  cover-glass.  Focus  the 
dust  carefully  first  with  the  low  then  with  the  high  objective.  When 
the  objective  is  in  focus  push  the  wedge  under  the  objective  on  the  slide 
until  it  touches  the  objective.  Mark  the  place  of  contact  with  a  pencil 
and  then  measure  the  thickness  of  the  wedge  with  a  rule  opposite  the 
point  of  contact.  This  thickness  will  represent  very  closely  the  work- 
ing distance.  For  measuring  the  thickness  of  the  wedge  at  the  point  of 
contact  for  the  high  objective  use  a  steel  scale  ruled  in  ^ths  mm.  and 
the  tripod  to  see  the  divisions.  Or  one  may  use  a  cover-glass  measurer, 
for  determining  the  thickness  of  the  wedge  (Ch.  VIII). 

For  the  higher  powers,  if  one  has  a  microscope  in  which  the  fine  ad- 
justment is  graduated,  the  working  distance  may  be  readily  determined 
when  the  thickness  of  the  cover-glass  over  the  specimen  is  known,  as 
follows  :     Get  the  object  in  focus,  lower  the  tube  of  the  microscope  un- 


CH,  II]  LIGHTING  AND  FOCUSING.  39 

til  the  front  of  the  objective  just  touches  the  cover-glass.  Note  the  po- 
sition of  the  micrometer  screw  and  slowly  focus  up  with  the  fine  ad- 
justment until  the  object  is  in  focus.  The  distance  the  objective  \\ 
raised  plus  the  thickness  of  the  cover-glass  represents  the  working  dis- 
tance. For  example,  a  3  mm.  objective  after  being  brought  in  contact 
with  a  cover-glass  was  raised  by  the  fine  adjustment  a  distance  repre- 
sented by  16  of  the  divisions  on  the  head  of  the  micrometer  screw. 
Each  division  represented  .01  mm.,  consequently  the  objective  was 
raised  .  16  mm.  As  the  cover-glass  on  the  specimen  used  was  .  15  mm. 
the  total  working  distance  is. 16  +.15  =.31  mm. 

CENTRAL    AND    OBLIQUE   LIGHT    WITH    A    MIRROR. 

§  73.  Axial  or  Central  Light  (§61). — Remove  the  condenser  or 
any  diaphragm  from  the  substage,  then  place  a  preparation  containing 
minute  air  bubbles  under  the  microscope.  The  preparation  may  he 
easily  made  by  beating  a  drop  of  mucilage  on  a  slide  and  covering  it. 
(See  Ch.  III).  Use  a  3  mm.  (JHj  in.)  or  No.  7  objective  and  a  medi- 
um ocular.  Focus  the  microscope  and  select  a  very  small  bubble,  one 
whose  image  appears  about  1  mm.  in  diameter,  then  arrange  the  plane 
.mirror  so  that  the  light  spot  in  the  bubble  appears  exactly  in  the  center. 
Without  changing  the  position  of  the  mirror  in  the  least,  replace  the  air- 
bubble  preparation  by  one  of  Plcurasigma  angulatiun  or  some  other 
finely  marked  diatom.     Study  the  appearance  very  carefully. 

§  74.  Oblique  Light,  (§  62). — Swing  the  mirror  far  to  one  side  so 
that  the  rays  reaching  the  object  ma)'  be  very  oblique  to  the  optic  axis 
of  the  microscope.  Study  carefully  the  appearance  of  the  diatom  with 
the  oblique  light.  Compare  the  different  appearance  with  that  of  cen- 
tral light.  The  effect  of  oblique  light  is  not  so  striking  with  histologi- 
cal preparations  as  with  diatoms. 

It  should  be  especially  noted  in  ££  73,  74,  that  one  cannot  deter- 
mine the  exact  direction  of  the  rays  by  the  position  of  the  mirror.  This 
is  especially  true  for  axial  light  (§  73).  To  be  certain  that  the  light  is 
axial  some  such  test  as  that  given  in  §  73  should  be  applied.  (See 
also  Ch.  Ill,  under  Air-bubbles). 

CONDENSERS    OR    ILLUMINATORS.* 

§  75.  These  are  lenses  or  lens-systems  for  the  purpose  of  illuminating 
with  transmitted  light  the  object  to  be  studied  with  the   micro-cope. 


*  No  one  has  stated  more  clearly  or  appreciated  more  truly  the  value  of  correct 
illumination  and  the  methods  of  obtaining  it  than  Sir  David  Brewster,  1820,  1831. 


40  LIGHTING  AND  FOCUSING.  [CH.  II 

For  the  highest  kind  of  investigation  their  value  cannot  be  overesti- 
mated. They  may  be  used  either  with  natural  or  artificial  light,  and 
should  be  of  sufficient  numerical  aperture  to  satisfy  objectives  of  the 
widest  angle. 

It  is  of  the  greatest  advantage  to  have  the  sub-stage  condenser 
mounted  with  rack  and  pinion  so  that  it  may  be  easily  moved  up  or 
down  under  the  stage.  The  iris  diaphragm  is  so  convenient  that  it 
should  be  furnished  in  all  cases,  and  there  should  be  marks  indicating 
the  N.  A.  of  the  condenser  utilized  with  different  openings.  Finally, 
the  condenser  should  be  supplied  with  central  stops  for  dark-ground 
illumination  (§  88)  and  with  blue  and  neutral  tint  glasses  to  soften  the 
glare  when  artificial  light  is  used  (§  85,  89). 

Condensers  or  Illuminators  fall  into  two  great  groups,  the  Achro- 
matic, giving  a  large  aplanatic  cone,  and  Non-achromatic,  giving 
much  light,  but  a  relatively  small  aplanatic  cone  of  light. 

§  76.  Achromatic  Condenser. — It  is  still  believed  by  all  expert  mi- 
croscopists  that  the  contention  of  Brewster  was  right,  and  the  condenser 
to  give  the  greatest  aid  in  elucidating  microscopic  structure  must  ap- 
proach in  excellence  the  best  objectives.  That  is,  it  should  be  as  free  as 
possible  from  spherical  and  chromatic  aberration,  and  therefore  would 
transmit  to  the  object  a  very  large  aplanatic  cone  of  light.  Such  con- 
densers are  especially  recommended  for  photo-micrography  by  all,  and 
those  who  believe  in  getting  the  best  possible  image  in  every  case  are 
equally  strenuous  that  achromatic  condensers  should  be  used  for  all 
work.  Unfortunately  good  condensers  like  good  objectives  are  expen- 
sive, and  student  microscopes  as  well  as  many  others  are  mostly  supplied 
with  the  non-achromatic  condensers  or  with  none. 

Many  excellent  achromatic  condensers  have  been  made,  but  the  most 
perfect  of  all  seems  to  be  the  apochromatic  of  Powell  and  Zealand  (Car- 
He  says  of  illumination  in  general  :  "The  art  of  illuminating  microscopic  objects 
is  not  of  less  importance  than  that  of  preparing  them  for  observation."  "The  eye 
should  be  protected  from  all  extraneous  light,  and  should  not  receive  any  of  the 
light  which  proceeds  from  the  illuminating  center,  excepting  that  portion  of  it 
which  is  transmitted  through  or  reflected  from  the  object."  So  likewise  the  value 
and  character  of  the  substage  condenser  was  thoroughly  understood  and  pointed 
out  by  him  as  follows  :  "I  have  no  hesitation  in  saying  that  the  apparatus  for  illu- 
mination requires  to  be  as  perfect  as  the  apparatus  for  vision,  and  on  this  account 
I  would  recommend  that  the  illuminating  lens  should  be  perfectly  free  of  chro- 
matic and  spherical  aberration,  and  the  greatest  care  be  taken  to  exclude  all  ex- 
traneous light  both  from  the  object  and  from  the  eye  of  the  observer."  See  Sir 
David  Brewster's  Treatise  on  the  Microscope,  1837,  pp.  136,  138,  146,  and  the  Edin- 
burgh Journal  of  Science,  new  series,  No.  11  (1831),  p.  83. 


CII.  II]  LIGHTING  AND  FOCUSING.  41 

penter-Dallinger,  pp.  254,  263).     To  attain  the  best  that  was  possible 
many  workers  have  adopted  the  plan  of  using  objectives  as  condense 
A  special  substage  fitting  is  provided  with  the  proper  screw  and  the  obj 
tive  is  put  into  position,  the  front  lens  being  next  the  object.      As  will 
be  seen  bslow  I  ^  79-80),  the  full  aperture  of  an  objective  can  rarely  b 
used,  and  for  histological  preparations  perhaps  never,  so  that  an  obj 
tive  of  greater  equivalent  focus,  /.  e. ,  lower  power  is  used  than  the  one 
on  the  microscope.     It  is  much  more  convenient,  however,  to  have  a 
special  condenser  with  iris  diaphragm  or  special  diaphragms  so  that  one 
may  use  any  aperture  at  wall,  and  thus  satisfy  the  conditions  necessary 
for  lighting  different  objects  for  the  same  objective  and  for  lighting  with 
objectives  of  different  apertures.     An  excellent  condenser  of  this  form 
has  been  produced  by  Zeiss  (Fig.  41).     It  has  a  total  numerical  aper- 
ture of  1. 00,  and  an  aplanatic  aperture  of  N.  A.  0.65. 

Fig.  41.  Zeiss'  Achromatic  Conden- 
ser, c.s  c  s.  Centering  screws  for 
changing  the  position  of  the  condenser 
and  making  its  axis  continuous  with 
that  of  the  microscope.  A  segment  of 
the  condenser  is  cut  away  to  show  the 
combinations  of  tenses.  For  very  low 
pozuers  the  upper  lens  is  sometimes 
screwed  off.  There  is  an  iris  dia- 
phragm between  the  middle  and  lower  ^  ^hP  c-s 
combinations.  (Zeiss'  Catalog,  No. 
So) 

§  77.  Centering  the  Condenser.  —To  get  the  best  possible  illumina- 
tion for  bringing  out  in  the  clearest  manner  the  minute  details  of  a  mi- 
croscopic object  two  conditions  are  necessary,  viz.  :  The  principal  optic 
axis  of  the  condenser  must  be  continuous  with  that  of  the  microscope 
(see  frontispiece)  and  the  object  must  be  in  the  focus  of  the  condenser, 
i.  c,  at  the  apex  of  the  cone  of  light  given  by  the  condenser. 

The  centering  is  most  conveniently  accomplished  as  follows  although 
daylight  may  be  used  with  almost  equal  facility  :  The  object  is  placed 
on  the  stage  and  lighted  with  the  edge  or  face  of  the  flame  and  then  a 
very  small  diaphragm  is  put  below  the  condenser.  (If  the  Zeiss  achi 
matic  condenser  is  used,  the  diaphragm  of  the  Abbe  illuminator  serves 
for  this.  If  there  is  no  pin-hole  diaphragm  one  can  be  made  of  -•tifl, 
black  paper,  care  must  be  taken,  however,  to  make  the  opening  exact- 
ly central.  This  is  best  accomplished  by  putting  the  paper  disc  over 
the  iris  or  metal  diaphragm  and  then  making  the  hole  in  the  center  of 


42 


LIGHTING  AND  FOCUSING. 


[CH.  II. 


the  small  circle  uncovered  by  the  metal  diaphragm.  For  the  hole  a  fine 
needle  is  best).  If  now  the  condenser  is  lowered  or  racked  away  from 
the  objective  the  image  of  the  diaphragm  will  appear.  If  the  opening 
is  not  central  it  should  be  made  so  by  using  the  centering  screws  of  the 
condenser. 

A  better  plan  than  to  lower  the  condenser  to  focus  the  image  of  the 
diaphragm,  is  to  raise  the  body  of  the  microscope  slowly  with  the  coarse 
adjustment.  It  is  almost  impossible  to  make  apparatus  so  accurate  that 
two  parts  like  the  body  of  the  microscope  and  the  substage,  each  work- 
ing on  different  sliding  surfaces,  shall  continue  in  exactly  the  same  plane. 
So  one  will  find  that  if  the  condenser  be  accurately  centered  with  the 
condenser  lowered,  and  then  the  condenser  be  racked  up  close  to  the 
stage  and  the  image  of  the  diaphragm  opening  brought  again  into  focus 
by  racking  up  the  body  of  the  microscope,  it  will  not  be  found  accu- 
rate^ centered  in  most  cases.  For  this  reason  it  is  advised  that  the 
condenser  be  left  in  position  close  to  the  stage  and  the  tube  of  the  mi- 
croscope be  used  to  focus  the  diaphragm  exactly  as  in  ordinary  work. 


Fig.  42.  Shows  that  the  optic  axis  of 
the  condenser  does  not  coincide  with 
that  of  the  microscope.  (D).  Dia- 
phragm of  the  condenser  shown  at  one 
side  of  the  field  of  the  microscope. 

Fig.  43  Shores  the  diaphragm  {D) 
in  the  center  of  the  field  of  the  micro- 
scope, and  thus  the  coincidence  of  the 
axis  of  the  condenser  with  that  of  the 
microscope. 


§  78.  Centering  the  Image  of  the  Source  of  Illumination. — For 
the  best  results  it  is  not  only  necessary  that  the  condenser  be  properly 
centered,  but  that  the  object  to  be  studied  should  be  in  the  image  of 
the  source  of  illumination  and  that  this  should  also  be  centered  (Figs. 
44,  45).  After  the  condenser  itself  is  centered  the  iris  diaphragm  is 
opened  to  its  full  extent  or  the  diaphragm  carrier  turned  wholly  aside. 
The  condenser  is  then  racked  up  toward  the  objective  until  the  image  of 
the  flame  is  apparently  on  the  specimen.  If  this  cannot  be  accomplished 
the  relative  position  of  the  lamp  and  condenser  is  not  correct  and  should 
be  so  changed  that  the  image  of  the  edge  of  the  flame  is  sharply  defined. 
This  image  must  also  be  centered.  This  is  easily  accomplished  by 
manipulation  of  the  mirror  or,  if  a  lamp  is  used,  by  changing  the  posi- 
tion of  the  lamp  or  of  the  bull's  eye  (Figs.  34,  52). 


CH.  II] 


LIGHTING  AND  FOCUSING. 


43 


^  79.  Proper  Numerical  Aperture  of  the  Condenser. — As  stated 
above,  the  aperture  of  the  condenser  should  have  a  range  by  means  of 
properly  selected  diaphragms  to  meet  the  requirements  of  all  objectn 

from  the  lowest  to  those  of  the  highest  aperture.  It  is  found  in  prac- 
tice that  for  diatoms,  etc.,  the  best  images  are  obtained  when  the  object 
is  lighted  with  a  cone  which  shall  fill  about  three-fourths  of  the  diameter 
of  the  back  lens  of  the  objective  with  light,  but  for  histological  and 
other  preparations  of  lower  refractive  power  only  one-half  or  one-third 
the  aperture  can  be  utilized. 


FlG.  44  Shows  the  image  of  the 
flame  (Fl.)  in  the  center  (C)  of  the 
field  of  the  microscope  and  illuminat- 
ing the  object. 

Fig.  45.  Shores  the  image  of  the 
flame  (Fl.)  at  one  side  of  the  center 
(F.\-c.)  and  not  properly  illuminating 
the  object. 


E  .<  i 


Fig.  44.  Fig.  45. 

To  determine  this  in  any  case,  focus  the  object  carefully,  take  out  the 
ocular,  look  down  the  tube  at  the  back  lens.  If  less  than  three-fourths 
of  the  back  lens  is  lighted,  increase  the  opening  in  the  diaphragm — if 
more  than  three-fourths,  diminish  it.  For  some  objects  it  is  advanta- 
geous to  use  the  entire  aperture,  for  others,  less  than  three-fourths. 
Experience  will  teach  the  best  lighting  for  special  cases. 


Obj 


Obj 


11 1  u 


1 1  l  urn 


Fig.  46. 


Fig.  47- 


Figs.  46-47.  Figures  showing  the  dependence  of  the  objective  upon  the  illumi- 
nating cone  of  the  condenser.     ( Nelson.) 

Fig.  46  (A).  The  illuminating  cone  f/om  the  condenser  (Ilium).  This  is 
seen  to  be  just  sufficient  to  fill  the  objective  (Obj. ). 

(  B).  The  back  lens  of  the  objective  entirely  Jilted  with  light,  showing  that  the 
numerical  aperture  of  the  illuminator  is  equal  to  that  of  the  objective. 

FlG.  47.  (A).  In  this  figure  the  illuminating  cone  from  the  condenser  \  Ilium.) 
is  seen  to  be  insufficient  to  Jill  the  objective  >  Obj. ). 

(A'i  The  back  lens  of  the  objective  only  partly  filled  with  light,  due  to  the  re- 
stricted aperture  of  the  illuminator. 


44  LIGHTING  AND  FOCUSING.  [CH.  II. 

§  80.  Aperture  of  the  Illuminating  Cone  and  the  Field. — It  is 
to  be  remarked  that  with  a  very  small  source  of  light  the  entire  aper- 
ture of  the  objective  may  ba  filled  if  a  proper  illuminator  or  condenser 
is  used.  The  aperture  depends  on  the  diaphragm  used  with  the  con- 
denser. And  the  size  of  the  diaphragm  must  be  directly  as  the  aper- 
ture of  the  objective.  That  is,  it  is  just  the  reverse  of  the  rule  for 
diaphragms  where  no  condenser  is  used  (§63)  ;  for  there  the  diaphragm 
is  made  large  for  low  powers,  and  consequently  low  apertures,  while 
with  the  condenser  the  diaphragm  is  made  small  for  low  and  large  for 
high  powers  as  the  aperture  is  greater  in  the  high  powers  of  a  given 
series  of  objectives.  It  is  very  instructive  to  demonstrate  this  by  using 
a  16  mm.  objective  and  opening  the  diaphragm  of  the  condenser  till  the 
bdck  lens  is  just  filled  with  light.  Then  if  one  uses  a  3  or  4  mm.  ob- 
jective it  will  be  seen  that  the  back  lens  of  the  higher  objective  is  only 
partly  filled  with  light,  and  to  fill  it  the  diaphragm  must  be  much  more 
widely  opened. 

With  a  condenser,  then,  the  diaphragm  has  simply  to  regulate  the 
aperture  of  the  illuminating  cone,  and  has  nothing  to  do  with  lighting 
a  large  or  a  small  field. 

With  the  condenser,  there  are  two  conditions  that  must  be  fulfilled, — 
the  proper  aperture  must  be  used,  and  that  is  determined  by  the  dia- 
phragm, and  secondly  the  whole  field  must  be  lighted.  The  latter  is  ac- 
complished by  using  a  larger  source  of  light,  as  the  face  instead  of  the 
edge  of  a  lamp  flame,  or  by  lowering  or  raising  the  condenser  so  that 
the  object  is  not  in  the  focus  of  the  condenser,  but  above  or  below  it, 
and  therefore  lighted  by  a  converging  or  diverging  beam  where  the 
light  is  spread  over  a  greater  area  (Figs.  48-51). 

§  81.  Non- Achromatic  Condenser. — Of  the  non-achromatic  con- 
densers or  illuminators,  the  Abbe  condenser  or  illuminator  is  the  one 
most  generally  used.  It  is  also  much  more  commonly  used  than  the 
achromatic  condenser  from  its  cheapness.  It  consists  of  two  or  three 
very  large  lenses  and  transmits  a  cone  of  light  of  1.20  N.  A.  to  1.40  N. 
A.,  but  the  aberrations,  both  spherical  and  chromatic,  are  very  great  in 
both  forms.  Indeed,  so  great  are  they  that  in  the  best  form  of  three 
lenses  with  an  illuminating  cone  of  1.40  N.  A.,  the  aplanatic  cone  trans- 
mitted is  only  0.5,  and  it  is  the  aplanatic  cone  which  is  of  real  use  in 
microscopic  illumination  where  details  are  to  be  studied.  There  is  no 
doubt,  however,  that  the  results  obtained  with  a  non-achromatic  con- 
denser like  the  Abbe  are  much  more  satisfactory  than  with  no  condenser. 
The  highest  results  cannot  be  attained  with  it,  however.  (Carpenter- 
Dallinger,  p.  256). 


CH.  //.]  LIGHTING  AND  FOCUSING.  45 

§  82.  Arrangement  of  the  Condenser. — The  proper  position  <■! 
the  illuminator  for  high  objectives  is  one  in  which  the  beam  of  li^ht 
traversing'  it  is  brought  to  a  focus  on  the  object.  If  parallel  rays  are 
reflected  from  the  plane  mirror  to  it,  they  will  be  focused  only  a  few 
millimeters  above  the  upper  lens  of  the  condenser  ;  consequently  the 
illuminator  should  be  about  on  the  level  of  the  top  of  the  stage  and 
therefore  almost  in  contact  with  the  lower  surface  of  the  slide.  For 
some  purposes,  when  it  is  desirable  to  avoid  the  loss  of  light  by  reflec- 
tion or  refraction,  a  drop  of  water  or  homogeneous  immersion  fluid  is 
put  between  the  slide  and  condenser,  forming  the  so-called  immersion 
illuminator.  This  is  necessary  only  with  objectives  of  high  power  and 
large  aperture  or  for  dark-ground  illumination. 

§  83.  Centering  the  Condenser.— The  illuminator  should  be  cen- 
tered to  the  optic  axis  of  the  microscope,  that  is  the  optic  axis  of  the 
condenser  and  of  the  microscope  should  coincide.  Unfortunately  there 
is  extreme  difficulty  in  determining  when  the  Abbe  illuminator  is  cen- 
tered. Centering  is  approximated  as  follows  :  Put  a  pin-hole  diaphragm 
over  the  end  of  the  condenser  (Pig.  48) — that  is,  a  diaphragm  with  a 
small  central  hole — the  central  opening  should  appear  to  be  in  the  mid- 
dle of  the  field  of  the  microscope.  If  it  does  not,  the  condenser  should 
be  moved  from  side  to  side  by  loosening  the  centering  screws  until  it  is 
in  the  center  of  the  field.  In  case  no  pin-hole  diaphragm  accompanies 
the  condenser,  one  may  put  a  very  small  drop  of  ink,  as  from  a  pen- 
point,  on  the  center  of  the  upper  lens  and  look  at  it  with  the  microscope 
to  see  if  it  is  in  the  center  of  the  field.  If  it  is  not,  the  condenser  should 
be  adjusted  until  it  is.  When  the  condenser  is  centered  as  nearly  as 
possible  remove  the  pin-hole  diaphragm  or  the  spot  of  ink.  The  micro- 
scope and  illminator  axes  may  not  be  entirely  coincident  even  when  the 
center  of  the  upper  lens  appears  in  the  center  of  the  field,  as  there  may 
be  some  lateral  tilting  of  the  condenser,  but  the  above  is  the  best  the 
ordinary  worker  can  do,  and  unless  the  mechanical  arrangements  of  the 
illuminator  are  very  deficient,  it  will  be  very  nearly  centered. 

It  is  to  be  hoped  that  the  opticians  will  devise  some  kind  of  mount- 
ing for  this  the  most  commonly  used  condenser  whereby  it  may  he  cen- 
tered as  described  for  the  achromatic  condenser  instead  of  by  the  crude 
methods  described  above.  If  the  condenser  mounting  regularly  p 
sessed  centering  screws  as  in  the  microscope  of  Watson  6c  S<mi>  1 
71  ),  and  there  was  a  centering  diaphragm  in  the  proper  position  -.1  that 
its  image  could  be  projected  into  the  field  of  view,  the  operation  would 
be  very  simple.  If,  further,  the  condensers  of  Powell  and  I.e.iland 
were  selected  as  models  the  condensers  need  not  be  SO  bulky,  and  still 
retain  all  their  efficiency. 


46  LIGHTING  AND  FOCUSING.  \_CH.  II. 

§  84.  Mirror  and  Light  for  the  Abbe  Condenser. — It  is  best  to 
use  light  with  parallel  rays.  The  rays  of  daylight  are  practically  par- 
allel ;  it  is  best,  therefore,  to  employ  the  plane  mirror  for  all  but  the 
lowest  powers.  If  low  powers  are  used  the  whole  field  might  not  be 
illuminated  with  the  plane  mirror  when  the  condenser  is  close  to  the  ob- 
ject ;  furthermore,  the  image  of  the  window  frame,  objects  outside  the 
building,  as  trees,  etc.,  would  appear  with  unpleasant  distinctness  in  the 
field  of  the  microscope.  To  overcome  these  defects,  one  can  lower  the 
condenser  and  thus  light  the  object  with  a  diverging  cone  of  light,  or 
use  the  concave  mirror  and  attain  the  same  end  when  the  condenser  is 
close  to  the  object  (Fig.  48). 

§  85.  Artificial  Light. — If  one  uses  lamplight,  it  is  recommended 
that  a  large  bull's  eye  be  placed  in  such  a  position  between  the  light 
and  the  mirror  that  parallel  rays  fall  upon  the  mirror  or  in  some  cases 
an  image  of  the  lamp  flame.  If  one  does  not  have  a  bull's  eye  the  con- 
cave mirror  may  be  used  to  render  the  rays  less  divergent.  It  may  be 
necessary  to  lower  the  illuminator  somewhat  in  order  to  illuminate  the 
object  in  its  focus. 

ABBE    CONDENSER  :    EXPERIMENTS. 

§  86.  Abbe  Condenser,  Axial  and  Oblique  Light. — Use  a  dia- 
phragm a  little  larger  than  the  front  lens  of  the  3  mm.  (}£  in.)  objec- 
tive, have  the  illuminator  on  the  level,  or  nearly  on  the  level,  of  the  up- 
per surface  of  the  stage,  and  use  the  plane  mirror.  Be  sure  that  the 
diaphragm  carrier  is  in  the  notch  indicating  that  it  is  central  in  position. 
Use  the  Pleurasigma  as  object.  Study  carefully  the  appearance  of  the 
diatom  with  this  central  light,  then  make  the  diaphragm  eccentric  so  as 
to  light  with  oblique  light.  The  differences  in  appearance  will  probably 
be  even  more  striking  than  with  the  mirror  alone  (§  74). 

§  87.  Lateral  Swaying  of  the  Image. — Frequently  in  studying  an 
object,  especially  with  a  high  power,  it  will  appear  to  sway  from  side  to 
side  in  focusing  up  or  down.  A  glass  stage  micrometer  or  fly's  wing 
is  an  excellent  object.  Make  the  light  central  or  axial  and  focus  up 
and  down  and  notice  that  the  lines  simply  disappear  or  grow  dim.  Now 
make  the  light  oblique,  either  by  making  the  diaphragm  opening  ec- 
centric or  if  simply  a  mirror  is  used,  by  swinging  the  mirror  sidewise. 
On  focusing  up  and  down,  the  lines  will  sway  from  side  to  side.  What 
is  the  direction  of  apparent  movement  in  focusing  down  with  reference 
to  the  illuminating  ray  ?  What  in  focusing  up  ?  If  one  understands 
this  experiment  it  may  sometimes  save  a  great  deal  of  confusion.  (See 
under  testing  the  microscope  for  swaying  with  central  light  §  104). 


CH.  //.] 


LIGHTING  AND  FOCUSING 


47 


§  88.    Dark-Ground  Illumination.-— When  an  object  is  Lighted  with 
rays  of  a  greater  obliquity  than  can  get  into  the  front  U  as  of  the  ol 
tive,  the  field  will  appear  dark  i  Pig.  51  ).     If  now  the  object  is  com 


°h-AU 


48. 


49- 


50. 


5'- 


Figs.  48-51.  Sectional  views  of  the  Abbe  Illuminator  of  1.20  N.  A.  showing 
various  methods  of  illumination  (g  S4).  Fig.  48,  axial  light  with  parallel  rays. 
Fig.  49,  oblique  light.  Fig.  50,  axial  light  with  converging  beam.  Fig.  51,  dark- 
ground  illumination  with  a  central  slop  diaphragm. 

Axis.  The  optic  axis  of  the  illuminator  and  of  the  microscope.  The  illumina- 
tor is  centered,  that  is  its  optic  axis  is  a  prolongation  of  the  optic  axis  of  the 
m  icroscope. 

S.  Axis.  Secondary  axis.  In  oblique  light  the  central  ray  passes  along  a  sec- 
ondary axis  of  the  illuminator,  and  is  therefore  oblique  to  the  principal  a.\  is. 

D  D.  Diaphragms.     These  are  placed  in  sectional  and  in  face  views.     The  dia- 
phragm is  placed  between  the  minor  and  the  illuminator.     In  Fig.  /y  the  open: 
is  eccentric  for  oblique  light,  and  in  Fig.  5/  the  opening  is  a  nan  the 

central  part  beitig  stopped  out,  and  thus  giving  rise  to  dark-ground  illumination 

Obj.  Obj.   The  front  of  the  objective. 

posed  of  fine  particles,  or  is  semi-transparent,  it  will  refract  or  reflect 
the  light  which  meets  it,  in  such  a  way  that  a  part  of  the-  very  oblique 
rays  will  pass  into  the  objective,  hence  as  light  readies  the  objective 
only  from  the  object,  all  the  surrounding  field  will  be  dark  and  the  ob 
ject  will  appear  like  a  self-luminous  one  on  a  dark  back-ground.     This 


48  LIGHTING  AND  FOCUSING.  [CH.  II. 

form  of  illumination  is  only  successful  with  low  powers  and  objectives 
of  small  aperture.  It  is  well  to  make  the  illuminator  immersion  for 
this  experiment,  see  §  98. 

(A)  With  the  Mirror — Remove  all  the  diaphragms  so  that  very 
oblique  light  may  be  used,  employ  a  stage  micrometer  in  which  the 
lines  have  been  filled  with  graphite,  use  a  16  mm.  (^3  in.)  objective, 
and  when  the  light  is  sufficiently  oblique  the  lines  will  appear  some- 
thing like  streaks  of  silver  on  a  black  back-ground.  A  specimen  like 
that  described  below  in  (B)  may  also  be  used. 

(B)  With  the  Abbe  Condenser. — Have  the  illuminator  so  that  the 
light  would  be  focused  on  the  object  (see  §  82)  and  use  a  diaphragm 
with  the  annular  opening  (Fig.  51)  ;  employ  the  same  objective  as  in 
(A).  For  object  place  a  drop  of  10  %  solution  of  salicylic  acid  in  95  r/o 
alcohol  on  the  middle  of  a  slide  and  allow  it  to  dry  and  crystallize.  The 
crystals  will  appear  brilliantly  lighted  on  a  dark  back-ground.  Put  in 
an  ordinary  diaphragm  and  make  the  light  oblique  by  making  the  dia- 
phragm eccentric.  The  same  specimen  may  also  be  tried  with  a  mir- 
ror and  oblique  light.  In  order  to  appreciate  the  difference  between 
this  dark-ground  and  ordinary  transmitted-light  illumination,  use  an 
ordinary  diaphragm  and  observe  the  crystals. 

A  very  striking  and  instructive  experiment  maj^  be  made  by  adding  a 
very  small  drop  of  the  solution  to  the  dried  preparation,  putting  it  under 
the  microscope  very  quickly,  lighting  for  dark-ground  illumination  and 
then  watching  the  crystallization. 

ARTIFICIAL   ILLUMINATION. 

§  89.  For  evening  work  and  for  regions  where  daylight  is  not  suf- 
ficiently brilliant,  artificial  illumination  must  be  employed.  Further- 
more, for  the  most  critical  investigation  of  bodies  with  fine  markings 
like  diatoms,  artificial  light  has  been  found  superior  to  daylight. 

A  petroleum  (kerosene)  lamp  with  flat  wick  gives  a  satisfactory  light. 
It  is  recommended  that  instead  of  the  ordinary  glass  chimney  one  made 
of  metal  with  a  slit-like  opening  covered  with  an  oblong  cover-glass  is 
more  satisfactory,  as  the  source  of  light  is  more  restricted.  Very  ex- 
cellent results  may  be  obtained,  however,  with  the  ordinary  bed-room 
lamp  furnished  with  the  usual  glass  chimney. 

The  new  acetylene  light  promises  to  be  the  most  perfect  of  all  the 
artificial  lights  for  microscopic  observation  and  for  photo-micrography. 
(See  under  Photo-micrography). 


CH.  //.] 


LIGHTING  AND  FOCUSING 


49 


Fig.  52.  1.  Lamp  with  slit-opening  in  metal  chimney.  2.  Bull's  eye  on  separate 
stand.     3.  Screen  showing  image  of  flame. 

Whenever  possible,  the  edge  of  the  flame  is  turned  toward  the  micro- 
scope, the  advantage  of  this  arrangement  is  the  greater  brilliancy,  due 
to  the  greater  thickness  of  the  flame  in  this  direction. 

£  90.  Mutual  Arrangement  of  Lamp,  Bull's  Eye  and  Micro- 
scope.— To  fulfill  the  conditions  given  above,  namely,  that  the  object 
be  illuminated  by  the  image  of  the  source  of  illumination  the  lamp 
must  Ik-  in  such  a  position  that  the  condenser  projects  a  sharp  image  of 
the  flame  upon  the  object  (Fig.  52),  and  only  by  trial  can  this  position 
be  determined.  In  some  cases  it  is  found  advantageous  to  discard  the 
mirror  and  allow  the  light  from  the  bull's  eye  to  pass  directly  into  the 
condenser.  This  method  is  especially  excellent  in  photomicrography 
(see  Ch.  YIII). 

§91,  Illuminating  the  Entire  Field.  —  With  low  objectives  and 
large  objects,  the  entire  "object  might  not  be  illuminated  if  the  above 
method  were  strictly  followed  ;  in  this  case,  turn  the  lamp  so  that  the 
flame  is  oblique,  or  if  that  is  not  sufficient,  continue  to  turn  the  lamp 
until  the  full  width  of  the  flame  is  used.  If  necessary  the  condenser 
may  be  lowered,  and  the  concave  mirror  used.      (See  also  >J  80 

REFRACTION    AND    COLOR    IMAGES. 

£  92.  Refraction  Images  are  those  mostly  seen  in  studying  microscopic  obj< 
They  are  the  appearances  produced  by  the  refraction  of  the  light  <>n  entering  ami 
on  leaving  an  object.     They  therefore  depend     .1    on  the  form  of  the  objei 
mi  tin-  relative  refractive  powers  of  object   and  mounting  medium.     With  such 
images  the  diaphragm  should  not  lie  too  large    see     79). 

If  the  color  and  refractive  index  of  the  object  were  exactly  like  the  mounting 
medium  it  could  not  he  seen.     In  mo-,t  cases  both  refractive  index  and  color  differ 
somewhat,  there  is  then  a  combination  of  color  ami  refraction  images  which  i>  a 
1 


5o 


LIGHTING  AND  FOCUSING. 


[CH.  II. 


great  advantage.     This  combination  is  generally  taken  advantage  of  in  histology. 
Fig.  89  is  an  example  of  a  purely  refractive  image. 


53.     N>.  54.     N',  55.     N'. 

Figs.  53-55. — Diagrams  illustrating  refraction  in  different  media  and  at  plane 
and  curved  surfaces.  In  each  case  the  denser  medium  is  represented  by  line  shad- 
ing and  the  perpendicular  or  normal  to  the  refracting  surface  is  represented  by  the 
dotted  line  N-N',  the  refracted  ray  by  the  bent  line  A  C. 

\  93.  Refraction. — Lying  at  the  basis  of  microscopical  optics  is  refraction,  which 
is  illustrated  by  the  above  figures.  It  means  that  light  passing  from  one  medium 
to  another  is  bent  in  its  course.  Thus  in  Fig.  53,  light  passing  from  air  into  water 
does  not  continue  in  a  straight  line  but  is  bent  toward  the  normal  N-N',  the 
bending  taking  place  at  the  point  of  contact  of  the  air  and  water  ;  that  is,  the  ray 
of  light  A  B  entering  the  water  at  B  is  bent  out  of  its  course,  extending  to  C  in- 
stead of  to  c. 

Conversely,  if  the  ray  of  light  is  passing  from  water  into  air,  on  reaching  the  air 
it  is  bent  from  the  normal,  the  ray  C  B  passing  to  A  and  not  in  a  straight  line  to 
Q." .  By  comparing  Figs.  54,  55,  in  which  the  denser  medium  is  crown  glass  in- 
stead of  water,  the  bending  of  the  rays  is  seen  to  be  greater  as  crown  glass  is  denser 
than  water. 

It  has  been  found  by  physicists  that  there  is  a  constant  relation  between  the  angle 
taken  by  the  ray  in  the  rarer  medium,  and  that  taken  by  the  ray  in  the  denser 
medium.  The  relationship  is  expressed  thus  :  Sine  of  the  angle  of  incidence  di- 
vided by  the  sine  of  the  angle  of  refraction  equals  the  index  of  refraction .  In 
Sin  A  BN 


the    figures, 


Sin  CBN' 


A  B  7V  =  4o°,  C  B  N'-- 


=  index  of  refraction.     Worked  out  completely  in  Fig.  53, 

28°  54/  and    Sin4°°      =  °<64279_  I-33    ,-.  e     the  index  of 
Sin  28°  54'       0.48327 

refraction  from  air  to  water  is  1.33.  (See  \  30).  In  Figs.  54-55,  illustrating  refrac- 
tion in  crown  glass,  the  angles  being  given,  the  problem  is  easily  solved  as  just 
illustrated.  ( For  table  of  natural  sines  see  third  page  of  cover;  for  interpolation 
see  p.  18,  \  29a). 

\  93  a.  Absolute  Index  of  Refraction. — This  is  the  index  of  refraction  ob- 
tained when  the  incident  ray  passes  from  a  vacuum  into  a  given  medium.  As  the 
index,  of  the  vacuum  is  taken  as  unity,  the  absolute  index  of  any  substance  is  al- 
ways greater  than  unity.  For  many  purposes,  as  for  the  purposes  of  this  book, 
air  is  treated  as  if  it  were  a  vacuum,  and  its  index  is  called  unity,  but  in  reality 
the  index  of  refraction  of  air  is  about  3  ten -thousandths  greater  than  unity. 
Whenever  the  refractive  index   of    a  substance   is  given,   the  absolute  index  is 


<'//.  //.]  LIGHTING  AND  FOCUSING  51 

meant  unless  otherwise  stated.     For  example,  when  the  ind<  \  of  refra<  tion  1 
ter  is  said  to  be  1 .33,  and  of  crown  glass  1.52,  etc.,  these  figures  represenl  the  ab- 
solute index,  and  the  incident  ray  is  supposed  to  be  in  a  vacuum. 

§93b.  Relative  Index  of  Refraction. — This  is  the  index  of  refraction  between 
two  contiguous  media,  .is  for  example  between  glassand  diamond,  water  and  gla 
etc.  It  is  obtained  by  dividing  the  absolute  index  of  refraction  of  the  substai 
containing  the  retracted  ray,  by  the  absolute  index  of  the  substance  transmitting 
the  incident  ray.  For  example,  the  relative  index  from  water  to  glass  is  [.52  di- 
vided by  [.33.  It'  the  Light  passed  from  glass  to  water  it  would  be,  t.33  divided  by 
1.52. 

By  a  study  of  the  figures  showing  refraction,  it  will  Ik-  seen  that  the  greater  the  re- 
fraction the  less  the  angle  and  consequently  the  less  the  sine  of  the  angle,  and  as  th< 
refraction  between  two  media  is  the  ratio  of  the  si  nes  of  the  angles  of  incidence  and  1 

fraction  \xx      )  it  will  be  seen  that  whenever  the  sine  of  the  angle  of  refraction  is 
\sin  // 

increased,  by  being  in  a  less  refractive  medium,  the  index  of  refraction  will  show 
a  corresponding  decrease  and  vice  versa.  That  is  the  ratio  of  the  sines  of  the 
angles  of  incidence  and  refraction  of  any  two  contiguous  substances  is  inversely  as 
the  refractive  indices  of  those  substances.     The  formula  is  : 

/  Sine  of  angle  of  incident  ray\      /  Index  of  refraction  of  refracting  medium  \ 
\Sine  of  angle  of  refracted  ray/     V  Index  of  refraction  of  incident  medium/ 

Abbreviated  I -: )=={  -. — s— — .  I.     By  means  of  this  general  formula  one  can  solve 

\  sin  r  J       \indcx    /  / 

any  problem  in  refraction  whenever  three  factors  of  the  problem  are  known.  The 
universality  of  the  law  may  be  illustrated  by  the  following  cases  : 

(A)   Light  incident  in  a  vacuum  or  in  air,  and  entering  some  denser  medium,  as 
water,  glass,  diamond,  etc. 

/  vSin  of  angle  made  by  the  ray  in  air  \       /Index  of  ref.  of  denser  nied.  \ 

\Sin  of  angle  made  by  ray  in   denser  medium/       \        Index  of  ref.  of  air     1  1       / 

If  the   dense  substance  were  glass:    I    .         I      |        -I.     If   the   two   media   were 

\snw7    V   >   / 

water  and  tdass,  the  incident  light  being  in  water  the  formula  would  be  :    I 

fe  5  6  ^Mn  rj 

(-  ) .    If  the  incident  rav  were  in  glass  and  the  refracted  ray  in  water  :    (    . 
'•33/  '  •  Vsin  r) 


(Hi)-  *■ 


d  similarly  for  any   two  media;  and   as  stated  above  if  any   three  of 


the  factors  are  given  the  fourth  may  readily  be  found. 

2  93  d.  Critical  Angle  and  Total  Reflection. — In  order  to  understand  the 
Wollaston  camera  lucida  ('i  171,  p.  11  1  1  and  other  totally  reflecting  apparatus,  it 
is  necessary  briefly  to  consider  the  critical  angle. 

The  critical  angle  is  the  greatest  angle  that  a  ray  of  light  in  the  denser  ••;  two 
contiguous  media  can  make  with  the  normal  and  still  emerge  into  the  1<  - 
tive  medium.     On  emerging  it   will  form  an  angle  of  900  with  the  normal,  and  if 
the  substance's  are  liquids,  the  refracted  ray  will  be  parallel  with  the  surfai  i  -a  the 
denser  medium. 

Total  Reflection. — In  case  the  incident  ray  in  the  denser  medium  is  at  an  angle 
with  the  normal,  greater  than  the  critical  angle,  it  will  be  totally  reflected  at  the 
surface  of  the  denser  medium,  that  surface  acting  as  a  perfect  mirror.  By  Consult- 
ing the  figures  it  will  be  seen  that  there  i-  u<>  sucb  thing  as  a  critical  angle  and 
total  reflection  in  the  rarer  of  two  contiguous  media  : 


52 


LIGHTING  AND  FOCUSING. 


\CH.  II. 


To  find  the  critical  angle  in  the  denser  of  two  contiguous  media  : — 

Make  the  angle  of  refraction   ( /.  e.,  the  angle  in  the  rarer  of  the  two  media) 

r,        -,  ,  .  ■  /sin  A      /index  r\ 

oo°  and  solve  the  general  equation  :   I  -.—     1=1.     ,         .  I.     Let  the  two  substances 

\sin  r)      \  index  i  J 

be  water  and  air,  then  the  sine  of  r  (900)  is  1,  the  index  of  air  is  1,  that  of  water 


/sin  i\      /     1     \ 
.33,   whence  I 


or  sin  i=  751  -f-     This  is  the  sine  of  48°  +  ,  and 


whenever  the  ray  in  the  water  is  at  an  angle  of  more  than  480  it  will  not  emerge 
into  the  air,  but  be  totally  reflected  back  into  the  water. 
The  case  of  a  ray  passing  from  crown  glass  into  water  : 
sin  i  \      /index  water  (1.33) ' 


(sin  i  \_('u 

sin  r  (sin  90°-=  1)/      \ii 


)'"t;'>m 


t sin  r  (sin  90°=  1 )/      \ index  glass  (1.52) 
whence  sin  i  =  .875  sine  of  critical  angle  in  glass  covered  with   water.     The  cor- 
responding angle  is  approximately  6i°. 

\  94.  Color  Images. — These  are  images  of  objects  which  are  strongly  colored, 
and  lighted  with  so  wide  an  aperture  that  the  refraction  images  are  drowned  in  the 
light.  vSuch  images  are  obtained  by  removing  the  diaphragm  or  by  using  a  larger 
opening.  This  method  of  illumination  is  specially  applicable  to  the  study  of 
stained  microbes.     (See  below,  \  101). 

ADJUSTABLE,    WATER    AND    HOMOGENEOUS    OBJECTIVES. 

EXPERIMENTS. 

\  95.  Adjustment  for  Objectives. — As  stated  above  {\  22),  the  aberration  pro- 
duced by  the  cover-glass  (Fig.  56),  is  compensated  for  by  giving  the  combinations 
in  the  objective  a  different  relative  position  than  they  would  have  if  the  objective 
were  to  be  used  on  uncovered  objects.  Although  this  relative  position  cannot  be 
changed  in  unadjustable  objectives,  one  can  secure  the  best  results  of  which  the 
objective  is  capable  by  selecting  covers  of  the  thickness  for  which  the  objective  was 
corrected.  (See  table  in  §27).  Adjustment  may  be  made  also  by  increasing  the 
tube-length  for  covers  tli inner  than  the  standard  and  by  shortening  the  tube-length 
for  covers  thicker  than  the  standard  (Fig.  57). 

Fig.  56. — Effect  of  the  cover-glass  on 
the  rays  from  the  object  to  the  objective 
( Ross ) . 

Axis.  The  projection  of  the  optic 
axis  of  the  microscope. 

F.  Focus  or  axial  point  of  the  objec- 
tive. 

F'  and  F" '.  Points  on  the  axis  where 
rays   2  and  3    appear   to   originate   if 
{raced  backward  after  emerging  from 
F  the  upper  side  of  the  cover-glass  ( Cover) . 

In  learning  to  adjust  objectives,  it  is  best  for  the  student  to  choose  some  object 
whose  structure  is  well  agreed  upon,  and  then  to  practice  lighting  it,  shading  the 
stage  and  adjusting  the  objective,  until  the  proper  appearance  is  obtained.  The 
adjustment  is  made  by  turning  a  ring  or  collar  which  acts  on  a  screw  and  increases 
or  diminishes  the  distance  between  the  systems  of  lenses,  usually  the  front  and  the 
back   systems    (Fig,    40). 

Ge?ieral  Directio7is. — (A)  The  thinner  the  cover-glass  the  further 
must  the  systems  of  lenses  be  separated,   i,  <?. ,   the  adjusting  collar  is 


3 

k   2 

1 

\v 

// 

•/ 

\  x  ^ 

y 

^ 

'  '  / 

\                 N 

■** 

•              / 

\                     N. 

\  ^ 

f 

,'   C  a  v  £  r 

n  N\ 

\^ 

, 

/  ■ 

\  ^  * 

SI 

\  \j 

"/     / 

\ . 

\r 

CII.  //.]  LIGHTING  AND  FOCUSING. 

turned  nearer  the  zero  or  the  mark  "  uncovered,"  and  conversely  ;  (B 
the  thicker  the  cover-glass  the  closer  together  are  the  systems  brought 
by  turning  the  adjusting  collar  from  the  zero  mark.     This  als<  i  Lnci 
the  magnification  of  the  objective  (Ch.  IV). 

The  following  specific  directions  for  making  the  cover-glass  adjust- 
ment are  given  by  Mr.  Wenham  (Carpenter,  166).     "Select  any  dark 
speck  or  opaque  portion  of  the  object,  and  bring  the  outline  into  pert' 
focus;  then  lay  the  finger  on  the  milled-head  of  the  fine  motion,  and 
move  it  briskly  backwards  and  forwards  in  both  directions  from  the  first 
position.     Observe  the  expansion  of  the  dark  outline  of  the  object,  both 
when  within  and  when  without  the  focus.     If  the  greater  expansion  ol- 
eoma is  when  the  object  is  without  the  focus,  or  farthest  from  the  ob- 
jective [/.  e. ,  in  focusing  up]  ,  the  lenses  must  be  placed  further  asunder, 
or  toward  the  mark  uncovered    [i.e.,  the   adjusting  collar   is  turned 
toward  the  zero  mark  as  the  cover-glass  is  too  thin  for  the  present  ad- 
justment].    If  the  greater  expansion  is  when  the  object  is  within  the 
focus,  or  nearest  the  objective,  [/.  c. ,  in  focusing  down]  ,  the  lenses  must 
be  brought  closer  together,  or  toward  the  mark  covered,  [z.  e.,  the  ad- 
justing collar  should  be  turned  away  from  the  zero  mark,  the  cover- 
glass  being  too  thick  for  the  present  adjustment]."     In  most  objectives 
the  collar  is  graduated  arbitrarily,  the  zero  (O)  mark  representing  the 
position  for  uncovered  objects.      Other  objectives  have  the  collar  graduated 
to  correspond  to  the  various  thickness  of  cover-glasses  for  which  the  object- 
ive may  be  adjusted.      This  seems  to  be  an  admirable  plan  ;   then  if  one 
knows  the  thickness  of  the  cover-glass  on  the  preparation  {Ch.   VIII  )  the 
adjusting  collar  may  be  set  at  a  corresponding  mark,  and  one  will  feel 
confident  that  the  adjustment  will  be  approximately  correct.     It  is  then 
only  necessary  for  the  observer  to  make  the  slight  adjustment  to  compensate 
for  the  mounting  medium  or  any  variation  from  the  standard  length  of 
the  tube  of  the  microscope.     In  adjusting  for  variations  of  the  length  of 
the  tube  from  the  standard  it  should  be  remembered  that  :   (A)   If  the 
tube  of  the  microscope  is  longer  than  the  standard  for  which  the  ob- 
jective was  corrected,  the  effect  is  approximately  the  same  as  thicken 
ing  the  cover-glass,  and  therefore  the  systems  of  the  objective  must 
brought  closer  together,  i.  e.,  the  adjusting  collar  must  be  turned  azi  ay 
from  the  zero  mark.      (B)   If  the  tube  is  shorter  than  the  standard  tor 
which  the  objective  is  corrected,  the  effect  is  approximately  the  sam< 
diminishing  the  thickness  of  the  cover-glass,   and   the   systems   m 
therefore  be  separated  (Fig.  40). 

In  using  the  tube-length  for  cover  correction  Shorten  the  tube  tot 
too  thick  covers  and  Lengthen  the  tube  for  too  thin  cow 


54 


LIGHTING  AND  FOCUSING. 


\CH.  II. 


Object-b 

Object— a 

Fig.  57. — Figure  to  show  that  in  lengthening  the  tube  of  the  microscope  the  ob- 
ject must  be  brought  nearer  the  principal  focus  or  center  of  the  lens.  It  will  be  seen 
by  consulting  the  figure  that  in  shortening  the  tube  of  the  microscope  the  object 
must  be  removed  farther  from  the  center  of  the  lens.  By  consulting  the  figure 
showing  the  effect  of  the  cover-glass  {Fig.  56)  it  will  be  seen  that  the  effect  of  the 
cover  glass  is  to  bring  the  object  nearer  the  objective,  and  the  thicker  the  cover  the 
nearer  is  the  object  brought  to  the  objective.  As  shortening  the  tube  serves  to  re- 
move the  object,  it  neutralizes  the  effect  of  the  thick  cover,  and  if  the  cover  is  so 
thin  that  it  does  not  elevate  the  object  enough  for  the  corrections  of  the  objective, 
then  an  increase  in  the  tube-length  will  correct  the  defect. 

Furthermore,  whatever  the  interpretation  by  different  opticians  of 
what  should  be  included  in  "  tube-length,"  and  the  exact  length  in  mil- 
limeters, its  importance  is  very  great  ;  for  each  objective  gives  the  most 
perfect  image  of  which  it  is  capable  with  the  "  tube-length  "  for  which 
it  is  corrected,  and  the, more  perfect  the  objective  the  greater  the  ill- 
effects  on  the  image  of  varying  the  "  tube-length  "  from  this  standard. 
The  plan  of  designating  exactly  what  is  meant  by  "  tube-length,"  and 


CM.  //.]  LIGHTING  AND  FOCUSING. 

engraving  on  each  objective  the  "  tube-length  "  for  which  it  is  correct 
ed,  is  to  be  commended,   for  it  is  manifestly  difficult  for  each  worl 
with  the  microscope  to  find  out  for  himself  for  what  "tube-length' 
each  of  his  objectives  was  corrected.     I  See  appendix 

§  97.  Water  Immersion  Objectives. — Put  a  water  immersion  ob- 
jective in  position  (§  43)  and  the  fly's  wing  for  object  under  the  mi< 
scope.  Place  a  drop  of  distilled  water  on  the  cover-glass,  and  with  the 
coarse  adjustment  lower  the  tube  till  the  objective  dips  into  the  water, 
then  light  the  field  well  and  turn  the  fine  adjustment  one  way  and  the 
other  till  the  image  is  clear.  Water  immersions  are  exceedingly  con- 
venient in  studying  the  circulation  of  the  blood,  and  for  many  other 
purposes  where  aqueous  liquids  are  liable  to  get  on  the  cover-glass.  If 
the  objective  is  adjustable,  follow  the  directions  given  in  §  95. 

When  one  is  through  using  a  water  immersion  objective,  remove  it 
from  the  microscope  and  with  some  lens  paper  wipe  all  the  water  from 
the  front-lens.  Unless  this  is  done  dust  collects  and  sooner  or  later  the 
front-lens  would  be  clouded.  It  is  better  to  use  distilled  water  to  avoid 
the  gritty  substances  that  are  liable  to  be  present  in  natural  waters,  as 
these  gritty  particles  might  scratch  the  front-lens. 

HOMOGENEOUS    IMMERSION    OBJECTIVES  \    EXPERIMENTS. 

>j  98.  As  stated  above,  these  are  objectives  in  which  a  liquid  of  the 
same  refractive  index  as  the  front-lens  of  the  objective  is  placed  between 
the  front-lens  and  the  cover-glass. 

§  99.  Tester  for  Homogeneous  Liquid. — In  order  that  full  ad- 
vantage be  derived  from  the  homogeneous  immersion  principle,  the 
liquid  employed  must  be  truly  homogeneous.  To  be  sure  that  such  is 
the  case,  one  may  use  a  tester  like  that  constructed  by  the  Gundlach 
Optical  Co.,  then  if  the  liquid  is  too  dense  it  ma}-  be  properly  diluted 
and  vice  versa.  For  the  cedar  oil  immersion  liquid,  the  density  may 
be  diminished  by  the  addition  of  pure  cedar  wood  oil.  The  density 
may  be  increased  by  allowing  it  to  thicken  by  evaporation.  (See  H. 
L.  Smith,  Proc.  Amer.  Soc.  Mier.,  1885,  p.  83,  and  appendix  1. 

§  100.   Refraction  Images. — Put  a  2  mm.  |  /..th  in.  I  homogent 
immersion   objective  in   position,    employ    an    illuminator.      I  me 

histological   specimen    like  a    muscular   fiber  as  object,   make  the  dia- 
phragm opening  abmt  3  111:11.  in  dianister,  all   a  drop  of  the  homo- 
geneous immersion  liquid  and   focus  as  directed  in  ^  7".     The  obji 
will  be  clearly  seen  in  all  details  by  the  unequal  refraction  of  the  light 
traversing  it.     The  difference  in  color  between  it  and  the  surround;- 


56 


LIGHTING  AND  FOCUSING, 


\CH.  II. 


medium  will  also  increase  the  sharpness  of  the  outline.  If  an  air  bub- 
ble preparation  (§  73)  were  used,  one  would  get  pure,  refraction  images. 
§  ior.  Color  Images. — Use  some  stained  microbes,  as  Bacillus  tuber- 
culosis for  object.  Put  a  drop  of  the  immersion  liquid  on  the  cover- 
glass  or  the  front  lens  of  the  homogeneous  objective.  Remove  the  dia- 
phragms from  the  illuminator  or  in  case  the  iris  diaphragm  is  used,  open 
to  its  greatest  extent.  Focus  the  objective  down  so  that  the  immersion 
fluid  is  in  contact  with  both  the  front  lens  and  the  cover-glass,  then 
with  the  fine  adjustment  get  the  microbes  in  focus.  They  will  stand 
out  as  clearly  defined  colored  objects  on  a  bright  field. 


Fig.  58. — Screen  for  shading  the  microscope  and  the 
face  of  the  observer.  This  is  very  readily  constructed 
as  shotvn  in  the  figure  by  supporting  a  wire  in  a  disc 
of  lead,  iron,  or  heavy  wood.  The  screen  is  then  com- 
pleted by  hanging  over  the  bent  wire,  cloth  or  man  ilia 
paper  30  x  40  cm.  The  lower  edge  of  the  screen  should 
be  a  little  below  the  stage  of  the  microscope  and  the 
upper  edge  high  enough  to  screen  the  eyes  of  the  ob- 
server. 


§  102.  Shading  the  Object. — To  get  the 
clearest  image  of  an  object  no  light  should 
reach  the  eye  except  from  the  object.  A  hand- 
kerchief or  a  dark  cloth  wound  around  the  ob- 
jective will  serve  the  purpose.  Often  the  proper  effect  may  be  obtained 
by  simply  shading  the  top  of  the  stage  with  the  hand  or  with  a  piece  of 
bristol  board.  Unless  one  has  a  very  favorable  light  the  shading  of  the 
object  is  of  the  greatest  advantage,  especially  with  homogeneous  im- 
mersion objectives.  The  screen  (Fig.  58)  is  the  most  satisfactory 
means  for  this  purpose,  as  the  entire  microscope  above  the  illuminating 
apparatus  is  shaded. 

§  103.  Cleaning  Homogeneous  Objectives. — After  one  is  through 
with  a  homogeneous  objective,  it  should  be  carefully  cleaned  as  follows  : 
Wipe  off  the  homogeneous  liquid  with  a  piece  of  the  lens  paper  (§  107), 
then  if  the  fluid  is  cedar  oil,  wet  one  corner  of  a  fresh  piece  in  benzin 
and  wipe  the  front  lens  with  it.  Immediately  afterward  wipe  with  a 
dry  part  of  the  paper.  The  cover-glass  of  the  preparation  can  be 
cleaned  in  the  same  way.  If  the  homogeneous  liquid  is  a  glycerin  mixt- 
ure proceed  as  above,  but  use  water  instead  of  benzin  to  remove  the 
last  traces  of  glycerin. 


CH.  II.]  LIGHTING  AND  FOCUSING. 

CAK1-:    <>F    Till'.    MICROSCOPE. 

£  104.  The  microscope  should  be  handled  carefully  and  kept  perl 

ly  clean.     The  oculars  ami  objectives  should  never  be  allowed  to  fall. 

When  not  in  use  keep  it  in  a  place  as  free  as  possible  from  dust. 

All  parts  of  the  microscope  should  be  kept  free  from  Liquids,  especial- 
ly from  acids,  alkalies,  alcohol,  benzin,  turpentine  and  chloroform. 

£  105.    Care  of  the  Mechanical  Parts.— To  clean  the  mechanical 

parts  put  a  small  quantity  of  some  fine  oil  (olive  oil  or  liquid  vaselin 
and  benzin,  equal  parts),  on  a  piece  of  chamois  leather  or  on  the  lens 
paper,  and  rub  the  parts  well,  then  with  a  clean  dry  piece  of  the  cha- 
mois or  paper  wipe  off  most  of  the  oil.  If  the  mechanical  parts  are 
kept  clean  in  this  way  a  lubricator  is  rarely  needed.  Where  opposed 
brass  surfaces  "cut,"  i.  e.t  when  from  the  introduction  of  some  gritty 
material,  minute  grooves  are  worn  in  the  opposing  surfaces,  giving  a 
harsh  movement,  the  opposing  parts  should  be  separated,  carefully 
cleaned  as  described  above  and  any  ridges  or  prominences  scraped  down 
with  a  knife.  Where  the  tendency  to  "  cut  "  is  marked,  a  very  slight 
application  of  equal  parts  of  beeswax  and  tallow,  well  melted  together, 
serves  a  good  purpose. 

In  cleaning  lacquered  parts,  benzin  alone  answers  well,  but  it  should 
be  quickly  wiped  off  with  a  clean  piece  of  the  lens  paper.  Do  not  use 
alcohol  as  it  dissolves  the  lacquer. 

^  106.  Care  of  the  Optical  Parts. — These  must  be  kept  scrupu- 
lously clean  in  order  that  the  best  results  may  be  obtained. 

Glass  surfaces  should  never  be  touched  with  the  fingers,  for  that  will 
soil  them. 

The  glass  of  which  the  lenses  are  made  is  quite  soft,  consequently  it 
is  necessary  that  only  soft,  clean  cloths  or  paper  be  used  in  wiping  them. 

Whenever  an  objective  is  left  in  position  on  a  microscope,  or  when 
several  are  attached  by  means  of  a  revolving  nose-piece,  an  ocular 
should  be  left  in  the  upper  end  of  the  tube  to  prevent  dust  from  falling 
down  upon  the  back-lens  of  the  objective. 

S  107.   Lens  Paper. — The  so-called  Japanese  filter  paper,  which  from 
its  use  with  the  microscope,  I  have  designated  lens  paper,  has  been  us 
in  the  author's  laboratory  for  the  last  ten  years  for  cleaning  the  lens 
of  oculars  and  objectives,   and  especially  for  removing  the  fluid  used 
with  immersion  objectives.      Whenever  a  piece  is  used  once  it  is  thrown 
away.      It  has  proved  more  satisfactory  than  cloth  or  chamois,  becau 
dust  and  sand  are  not  present  ;  and  from  its  bibulous  character  it  is  very 
efficient  in  removing  liquid  or  semi-liquid  substances. 


53  LIGHTING  AND  FOCUSING.  [CH.  II. 

§  10S.  Dust  may  be  removed  with  a  camel's  hair  brush,  or  by  wiping 
with  the  lens  paper. 

Cloudiness  may  1)3  removed  from  the  glass  surfaces  by  breathing  on 
them,  then  wiping  quickly  with  a  soft  cloth  or  the  lens  paper. 

Cloudiness  on  the  inner  surfaces  of  the  ocular  lenses  may  be  removed 
by  unscrewing  them  and  wiping  as  directed  above.  A  high  objective 
should  never  be  taken  apart  by  an  inexperienced  person. 

If  the  cloudiness  cannot  be  removed  as  directed  above,  moisten  one  cor- 
ner of  the  cloth  or  paper  with  95  per  cent,  alcohol,  wipe  the  glass  first 
with  this,  then  with  the  dry  cloth  or  the  paper. 

Water  may  be  removed  with  soft  cloth  or  the  paper. 

Glycerin  may  be  removed  with  cloth  or  paper  saturated  with  distilled 
water  ;  remove  the  water  as  above. 

B'ood  or  othir  albumbuus  material  may  be  removed  while  fresh  with 
a  moist  cloth  or  paper,  the  same  as  glycerin.  If  the  material  has  dried 
to  the  glass,  it  may  be  removed  more  readily  by  adding  a  small  quan- 
tity of  ammonia  to  the  water  in  which  the  cloth  is  moistened,  (water 
ioocc,  ammonia  1  cc). 

Canada  Balsam,  damar,  paraffin,  or  any  oily  substance,  may  be  re- 
moved with  a  cloth  or  paper  wet  with  chloroform,  benzin  or  xylene. 
The  application  of  these  liquids  and  their  removal  with  a  soft,  dry  cloth 
or  paper  should  be  as  rapid  as  possible,  so  that  none  of  the  liquid  will 
have  time  to  soften  the  setting  of  the  lenses. 

Shellac  Cement  may  be  removed  by  the  paper  or  a  cloth  moistened  in 
95  per  cent,  alcohol. 

Brunswick  Black,  Gold  Size,  and  all  other  substances  soluble  in  chlo- 
roform, etc.,  may  be  removed  as  directed  for  balsam  and  damar. 

In  general,  use  a  solvent  of  the  substance  on  the  glass  and  wipe  it  off 
quickly  with  a  fresh  piece  of  the  lens  paper. 

It  frequently  happens  that  the  upper  surface  of  the  back  combination 
of  the  objective  becomes  dusty.  This  may  be  removed  in  part  by  a 
brush,  but  more  satisfactorily  by  using  a  piece  of  the  soft  paper  loosely 
twisted.  When  most  of  the  dust  is  removed  some  of  the  paper  may  be 
put  over  the  end  of  a  pine  stick  (like  a  match  stick )  and  the  glass  sur- 
face carefully  wiped. 

CARE    OF   THE    EYES. 

§  109.  Keep  both  eyes  open,  using  the  eye-screen  if  necessary  (Fig. 
59,  6d)  ;  and  divide  the  labor  between  the  two  eyes,  i.  e..  use  one  eye 
for  observing  the  image  awhile  and  then  the  other.     In  the  beginning 


CH.  II.} 


LIMITING  AND  FOCUSING. 


59 


it  is  not  advisable  to  look  into  the  microscope  continuously  for  more 

than  half  an  hour  at  a  time.  One 
never  should  work  with  the  micro- 
scope after  the  eyes  feel  fatigued. 
After  one  becomes  accustomed  to  mi- 
croscopic observation  he  tan  work  for 
pXG.  59._  Ward's  Eye-Shade.  several  hours  with  the  microscope 
without  fatiguing  the  eyes.  This  is  due  to  the  fact  that  the  eyes  be- 
come inured  to  labor  like  the  other  organs  of  the  body  by  judicious  ex 
ercise.  It  is  also  due  to  the  fact  that  but  very  slight  accommodation  is 
required  of  the  eyes,  the  eyes  remaining  nearly  in  a  condition  of  rest  as 
for  distant  objects.  The  fatigue  incident  upon  using  the  microscope  at 
first  is  due  parti}'  at  least  to  the  constant  effort  on  the  part  of  the  ob- 
server to  remedy  the  defects  of  focusing  of  the  microscope  by  accommo- 
dation of  the  eyes.  This  should  be  avoided  and  the  fine  adjustment  of 
the  microscope  used  instead  of  the  muscles  of  accommodation.  With  a 
microscope  of  the  best  quality,  and  suitable  light — that  is  light  which  is 
steady  and  not  so  bright  as  to  dazzle  the  eyes  nor  so  dim  as  to  strain 
them  in  determining  details — microscopic  work  should  improve  rather 
than  injure  the  sight. 

Fig.  6d.  Double  Eye  Shade.  This  is 
readily  made  by  taking  some  thick  bris- 
tol  board  7  x  ij  centimeters  and  making 
an  oblong  opening  with  rounded  ends 
(0—0)  and  of  such  a  diameter  that  it  goes 
readily  over  the  tube  of  the  microscope. 
This  is  then  covered  on  both  sides  with 
velveteen  and  a  central  slit  (s)  made  in 
the  cloth  This  ad in  its  the  tube  of  the 
microscope  and  holds  the  screen  in  posi- 
tion. It  may  readily  be  pulled  from  side  to  side  and  thus  serves  for  either  eye,  or 
for  the  use  of  the  eyes  alternately. 

^  1 10.  Position  and  Character  of  the  Work-Table. — The  work 
table  should  be  very  firm  and  large  (122  x  72  cm.  ;  28  x  48  in.),  so  that 
the  necessary  apparatus  and -material  for  work  may  not  be  too  crowded. 
The  table  should  also  be  of  the  right  height  to  make  work  by  it  com- 
fortable. An  adjustable  stool,  something  like  a  piano  stool  is  conven- 
ient, then  one  may  vary  the  height  corresponding  to  the  necessities  ol 
special  cases.  It  is  a  great  advantage  to  sit  facing  the  window  if  day- 
light is  used,  then  the  hands  do  not  constantly  interfere  with  the  illu- 
mination. To  avoid  the  discomfort  of  facing  the  light  a  screen  like  that 
shown  in  Fig.  5S  is  very  useful  1  see  also  under  lighting,  - 


7 

X 

14 

cm. 

, —  . 

/o 

1 

s 

0  \ 

I 

1 

/ 
/ 

60  LIGHTING  AND  FOCUSING.  [CII.  II. 

TESTING  THE   MICROSCOPE. 

\  in.  Testing  the  Microscope.— To  be  of  real  value  this  must  be  accomplished 
by  a  person  with  both  theoretical  and  practical  knowledge,  and  also  with  an  un- 
prejudiced mind.  Such  a  person  is  not  common,  and  when  found,  does  not 
show  au  over  anxiety  to  pass  judgment.  Those  most  ready  to  offer  advice  should 
as  a  rule  be  avoided,  for  in  most  cases  they  simply  "  have  an  ax  to  grind,"  and  are 
sure  to  commend  only  those  instruments  that  conform  to  the  "fad"  of  the  day. 
From  the  writer's  experience  it  seems  safe  to  say  that  the  inexperienced  can  do  no 
better  than  to  trust  to  the  judgment  of  one  of  the  optical  companies.  The  makers 
of  microscopes  and  objectives  guard  with  jealous  care  the  excellence  of  both  the 
mechanical  and  optical  part  of  their  work,  and  send  out  only  instruments  that  have 
been  carefully  tested  and  found  to  conform  to  the  standard.  This  would  be  done 
as  a  matter  of  business  prudence  on  their  part,  but  it  is  believed  by  the  writer  that 
microscope  makers  are  artists  first  and  take  an  artist's  pride  in  their  work,  they 
therefore  have  a  stimulus  to  excellence  greater  than  business  prudence  alone  could 
give. 

\  112.  Mechanical  Parts. — All  of  the  parts  should  be  firm,  and  not  too  easily 
shaken.  Bearings  should  work  smoothly.  The  mirror  should  remain  in  any  posi- 
tion in  which  it  is  placed. 

Focusing  Adjustments. — The  coarse  or  rapid  adjustment  should  be  by  rack  and 
pinion,  and  work  so  smoothly  that  even  the  highest  power  can  be  easily  focused 
with  it.  In  no  case  should  it  work  so  easily  that  the  body  of  the  microscope  is 
liable  to  run  down  and  plunge  the  objective  into  the  object.  If  any  of  the  above 
defects  appear  in  a  microscope  that  has  been  used  for  some  time,  a  person  with 
moderate  mechanical  instinct  will  be  able  to  tighten  the  proper  screws,  etc. 

The  Fine  Adjustment  is  more  difficult  to  deal  with.  From  the  nature  of  its 
purpose,  unless  it  is  approximately  perfect,  it  would  better  be  off  the  microscope 
entirely. 

It  should  work  smoothly  and  be  so  balanced  that  one  cannot  tell  by  the  feeling 
when  using  it  whether  the  screw  is  going  up  or  down.  Then  there  should  be  ab- 
solutely no  motion  except  in  the  direction  of  the  optic  axis,  otherwise  the  image 
will  appear  to  sway  even  with  central  light.  Compare  the  appearance  when  using 
the  coarse  and  when  using  the  fine  adjustment.  There  should  be  no  swaying  of 
the  image  with  either  if  the  light  is  central  {\  73). 

\  113.  Testing  the  Optical  Parts. — -As  stated  in  the  beginning,  this  can  be  done 
satisfactorily  only  by  an  expert  judge.  It  would  be  of  very  great  advantage  to  the 
student  if  he  could  have  the  help  of  such  a  person.  In  no  case  is  the  condemna- 
tion of  a  microscope  to  be  made  by  au  inexperienced  person.  If  the  beginner  will 
bear  in  mind  that  his  failures  are  due  mostly  to  his  own  lack  of  knowledge  and 
lack  of  skill  ;  and  will  truly  endeavor  to  learn  and  apply  the  principles  laid  down 
in  this  and  in  the  standard  works  referred  to,  he  will  learn  after  a  while  to  estimate 
at  their  true  value  all  the  pieces  of  his  microscope.     (See  appendix). 


CI  I.  II.  ]  /  .  /  //<  >A>.  I  TO A'y  .)//(  AV  >S<  ( >/  7  s 

LABORATORY  COMPOUND  MICROSCOPES. 

§  114.  Optical  Parts.— A  great  deal  of  beginning  work   with   the  mi  pe  in 

biological  laboratories  is  done  with  simple  and  inexpensive  apparatus,     [ndeed  if 
one  contemplates  the  large  classes  in  the  universities  and  inch.  it  can 

be  readily  understood  that  microscopes  costing  from  J  ad  magnifying 

from  25  to  500  diameters,  are  all  that  can  be  expected.  But  for  the  pm  ; 
modern  histological  investigation  and  of  advanced  microscopical  work  in  general, 
a  microscope  should  have  something  like  the  following  character  :  Its  optical  out- 
fit should  comprise,  (a)  dry  objectives  of  50  mm.  (2  in. ),  1 6-1 8  mm.  1  ;t  in.)  and  J 
mm.  1  \s  in.)  equivalent  focus.  There  should  be  present  also  a  2  mm.  ,:  in.  1  or 
1.5  mm.  (  rV,  in.)  homogeneous  immersion  objective.  Of  oculars  there  should  be 
several  of  different  power.  An  illuminator  or  substage  condenser,  and  an  A' 
camera  lucida  are  also  necessities,  and  a  micro-spectroscope  and  a  micro-polarizer 
are  very  desirable. 

Even  in  case  all  the  optical  parts  cannot  be  obtained  in  the  beginning,  it  is 
to  secure  a  stand  upon  which  all  may  be  used  when  they  are  finallv  secured. 

As  to  the  objectives.  The  best  that  can  be  afforded  should  be  obtained.  Cer- 
tainly at  the  present,  the  apochromatics  stand  at  the  head,  although  the  best  achro- 
matic objectives  approach  them  very  closely. 

\  115.  Mechanical  Parts  or  Stand. — The  stand  should  be  low  enough  so  that  it 
cm  be  used  in  a  vertical  position  on  an  ordinary  table  without  inconvenience  ;  it 
should  have  a  jointed  (flexible)  pillar  for  inclination  at  any  angle  to  the  horizontal. 
The  adjustments  for  focusing  should  be  two, — a  coarse  adjustment  or  rapid  move- 
ment with  rack  and  pinion,  and  a  fine  adjustment  by  means  of  a  micrometer  screw. 
Both  adjustments  should  move  the  entire  tube  of  the  microscope.  The  body  or 
tube  should  be  short  enough  for  objectives  corrected  for  the  short  or  160  millimi 
tube-length,  and  the  draw-tube  should  be  graduated  in  centimeters  and  millimeters. 
The  lower  end  of  the  draw  tube  and  of  the  tube  should  each  possess  a  standard 
screw  for  objectives  (frontispiece).  The  stage  should  be  quite  large  for  the  exami- 
nation of  slides  with  serial  sections.  If  there  is  no  mechanical  stage  >  '  1 
also  of  considerable  advantage  to  have  the  stage  with  a  circular,  revolving  top, 
and  two  centering  screws  with  milled  heads.  In  this  way  a  mechanical  stage  with 
limited  motion  is  secured,  and  this  is  of  the  highest  advantage  in  using  powerful 
objectives.  The  sub-stage  fittings  should  be  so  arranged  as  to  enable  one  to  dis- 
pense entirely  with  diaphragms,  to  use  ordinary  diaphragms,  or  to  use  the  conden- 
ser. The  condenser  mounting  should  allow  up  and  down  motion,  preferably  by 
rack  and  pinion.  The  base  should  be  sufficiently  heavy  and  so  arranged  that  the 
microscope  will  be  steady  in  all  positions,  and  interfere  the  least  possible  amount 
with  the  manipulation  of  the  mirror  and  other  sub-stage  accessories. 

\  116.   Mechanical  Stage. — There  should  also  be  present  some  form  of  mechani- 
cal stage.     That  on  the  most  expensive  American  and  English  tni<  1 
last  twenty  years  and  the  one  now  present  on  the  larger  continental   m 
is  excellent  for  high  powers  and  preparations  of  moderate  dimensions,  bi  I  foi   the 
study  of  serial  sections  and  large  sections  or  preparations  in  general,  m<  ■ 
stages  like  those  shown  in  Pigs.  68-69  are  more  useful.     This  form  oi  mei 
stage  has  the  advantage  of  giving  great  lateral  and  forward  and  bat  kward  mol 
It  is  a  modification  of  the  mechanical  sta  ;e  of  Toll.  e  modification  1 


62  LABORATORY  MICROSCOPES.  [CH.  II 

in  removing  the  thin  plate  and  instead,  having  a  elamp  to  catch  the  ends  of  the 
glass  slide.  The  slide  is  then  moved  on  the  face  of  the  stage  proper.  This  modi- 
fication was  first  made  by  Mayall.  It  has  since  been  modified  by  Reichert,  Zeiss, 
Leitz  and  others  in  Europe  and  by  the  Bausch  &  Lomb  Optical  Co.  in  America. — 
(Jour.  Roy.  Micr,  Soc,  1SS5,  p.  122.  See  also  Zeit.  wiss.  Mikroskopie,  (II),  1885, 
pp.  2S9  295  ;  18S7,  (IV,,  pp.  25-30). 

\  117.  Society  Screw. — Owing  to  the  lack  of  uniformity  in  screws  for  microscopic 
objectives,  the  Royal  Microscopical  Society  of  London,  in  1S57,  made  an  earnest 
effort  to  introduce  a  standard  size.  The  specifications  of  this  standard  are  as  fol- 
lows :  "  Whitvvorth  thread,  i  e.,  a  V  shaped  thread,  sides  of  thread  inclined  to  an 
angle  of  550  to  each  other,  one-sixth  of  the  V  depth  of  the  thread  being  rounded  off 
at  the  top  of  the  thread,  and  one-sixth  of  the  thread  being  rounded  off  at 
the  bottom  of  the  thread.  Pitch  of  screw,  36  to  the  inch  ;  length  of  thread  on 
object-glass,  0.125  inch  ;  plain  fitting  above  thread  of  object  glass,  o  15  inch  long, 
to  be  about  the  size  of  the  bottom  of  male  thread  ;  length  of  thread  of  nose-piece 
[on  the  lower  end  of  the  tube  of  the  microscope],  not  less  than  o  125  inch  ;  diam- 
eter of  the  object-glass  screw  at  the  bottom  of  the  screw,  o  7626  inch  ;  diameter 
of  the  nose  piece  screw  at  the  bottom  of  the  thread,  o  8  inch. 

In  order  to  facilitate  the  introduction  of  this  universal  screw,  or  as  it  soon  came 
to  be  called  "  The  Society  Screzu"  the  Royal  Microscopical  Society  undertook  to 
supply  standard  taps.  From  the  mechanical  difficulty  in  making  these  taps  per- 
fect there  soon  came  to  be  considerable  difference  in  the  "Society  Screws,"  and 
the  object  of  the  society  in  providing  a  universal  screw  was  partly  defeated. 

In  1S84  the  American  Microscopical  Society  appointed  Mr.  Edward  Bausch  and 
Prof.  William  A  Rogers  upon  a  committee  to  correspond  with  the  Royal  Micro- 
scopical Society,  with  a  view  to  perfecting  the  standard  "  .Society  Screw,"  or  of 
adopting  another  standard  and  of  perfecting  methods  by  which  the  screws  of  all 
makers  might  be  truly  uniform.  Although  this  matter  was  earnestly  considered  at 
the  time  by  the  Royal  Microscopical  Society,  the  mechanical  difficulties  were  so 
great  that  the  improvements  were  abandoned. 

Fortunately,  however,  during  the  present  year  (1896)  that  society  has  again 
taken  hold  of  the  matter  in  earnest,  and  we  are  now  promised  a  new  "  Societ}' 
Screw"  which  shall  be  accurate  ;  and  facilities  for  obtaining  the  standard  will  be 
so  good  that  there  is  a  reasonable  certainty  that  the  universal  screw  for  micro- 
scopic objectives  may  be  realized.  It  is  indeed  astonishing  to  see  how  widely 
spread  the  "  Society  Screw  "  has  become.  Indeed  there  is  not  a  maker  of  first 
clas-;  microscopes  in  the  world  who  does  not  supply  the  objectives  and  stands  with 
the  "  Society  Screw,"  and  an  objective  in  England  or  America  which  does  not  have 
this  screw  should  be  looked  upon  with  suspicion.  That  is,  it  is  either  old,  cheap, 
or  not  the  product  of  one  of  the  great  opticians.  For  the  Standard,  or  "Society 
Screw."  see:  Trans.  Roy.  Micr.  Soc,  1857,  pp.  39-41  ;  1859,  PP-  92"  97  !  1S60,  pp. 
103-104.  (All  to  be  found  in  Quar.  Jour.  Micr.  Sci.,  o.  5.,  vols.  VI,  VII  and  VIII). 
Proc.  Amer.  Micr.  Soc,  1884,  p.  274;  1886,  p.  199;  1893,  p.  38.  Journal  of  the 
Royal  Microscopical  Society,  Aug.,  1896. 

In  this  last  paper  of  four  pages  the  matter  is  very  carefully  gone  over  and  full 
specifications  of  the  new  screw  given.  It  conforms  almost  exactly  with  the  orig- 
inal standard  adopted  by  the  society,  but  means  have  been  devised  by  which  it  may 
be  kept  standard. 


CH.  II.  ]  LABOR  A  7  OR  > '  MICRi  >SCOI  '£  S  63 

FIGURES  OF   LABORATORY   MICROSCOPES  AND 
ACCESSORY   APPARATUS. 

It  was  deemed  advisable  in  this  new  edition  to  figure  some  of  the 
most  common  of  the  laboratory  microscopes  and  this  has  been  rendered 
possible  mostly  by  the  courtesy  of  the  makers  and  importers.  During 
the  last  five  years  very  great  vigor  has  been  shown  in  the  microscopical 
world.  This  has  been  stimulated  largely  by  the  activity  in  biological 
science  and  the  widespread  appreciation  of  the  microscope,  not  only  as 
a  desirable,  but  as  a  necessary  instrument  of  study  and  research.  The 
production  of  the  new  kinds  of  glass  (Jena  glass),  and  the  apochromatic 
objectives  have  been  a  no  less  potent  factor  in  promoting  progress.  It 
is  gratifying  also  to  know  that  with  the  increase  in  the  use  of  the  mi- 
croscope, not  only  are  the  optical  and  mechanical  parts  improved,  and 
that  very  greatly,  but  the  price  has  decreased  so  that  at  the  present  time 
schools  cannot  afford  to  be  without  one  or  more,  and  individuals  are  not 
debarred  from  the  possession  of  an  instrument  adequate  to  their  need-. 
The  cost  of  a  complete  outfit  varies  from  25  to  600  dollars.  The  stu- 
dent is  advised  to  write  to  one  or  more  of  the  opticians  for  complete 
catalogs.     See  list,  p.  2  of  cover. 

\  118.  Marker  tor  Preparations.  (Figs.  61-66). — This  instrument  consists  of  an 
objective-like  attachment  which  may  he  screwed  into  the  nose-piece  of  the  micro- 
scope. It  bears  on  its  lower  end  (  Figs.  61-3)  a  small  brush  and  the  brush  can  be 
made  more  or  less  eccentric  and  can  be  rotated,  thus  making  a  larger  or  smaller 
circle.  In  using  the  marker  the  brush  is  dipped  in  colored  shellac  or  other  cement 
and  when  the  part  of  the  preparation  to  be  marked  is  found  and  put  exactly  in  the 
middle  of  the  field  the  objective  is  turned  aside  and  the  marker  turned  into  posi- 
tion. The  brush  is  brought  carefully  in  contact  with  the  cover-glass  and  rotated. 
This  will  make  a  delicate  ring  of  the  colored  cement  around  the  ohjeet.  Within 
this  very  small  area  the  desired  object  can  be  easily  found  on  any  microscope. 
The  brush  of  the  marker  should  be  cleaned  with  95  %  alcohol  after  it  is  used. 
(Proc.  Amer,  Micr.  Soc,  1S94,  pp.  112-118). 

\  119.  Pointer  in  the  Ocular. — The  Germans  have  a  pointer  ocular  (Spitzen- 
Okular),  an  ocular  with  one  or  two  delicate  rods  or  pointers  at  the  level  of  the 
real  image,  that  is,  at  the  level  of  the  diaphragm  (Figs.  21,  30  I)).  For  the 
purposes  of  demonstrating  any  particular  structure  or  object  in  the  field,  a  tempo- 
rary pointer  may  be  easily  inserted  in  any  ocular  as  follows  :  Remove  the  eye-lens 
and  with  a  little  mucilage  or  Canada  balsam  fasten  an  eye  lash  (cilium  >  to  the 
diaphragm  (Fig.  30  D)  so  that  it  will  project  about  half  way  across  the  opening. 
If  one  uses  this  ocular,  the  pointer  will  appear  in  the  field  and  one  can  place  the 
specimen  so  that  the  pointer  indicates  it  exactly,  as  in  using  a  pointer  on  a  diagram 
or  on  the  black-board.  It  is  not  known  to  the  author  who  devised  this  method. 
It  is  certainly  of  the  greatest  advantage  in  demonstrating  obj«  eta  like  amoebas  or 
white  blool  corpuscles  to  persons  not  familiar  with  them,  as  the  field  is  liable  to 


64 


LA  BORA  TOR  Y  MICROSCOPES. 


[CH.  II. 


have  in  it  many  other  objects  which  are  more  easily  seen,  as  the  red  blood  corpus- 
cles or  particles  of  vegetation  or  dirt  in  the  case  of  the  blood  preparation  or  of  the 
amoeba. 


SS 


SS 


61.  62. 

Figs.  61-63.  Sectional  Views  of  the  two  Forms  of  the  Marker. 

Fig.  61.  The  simplest  form  of  marker.  It  consists  of  the  part  SS  with  the 
milled  edge  ( M ).  This  part  bears  the  Society  or  objective  screzufor  attaching  the 
marker  to  the  microscope.  R.  Rotating  part  of  the  marker.  This  bears  the  eccen- 
tric brush  (B)  at  its  lower  end.  The  brush  is  on  a  wire  (  W).  This  -wire  is  eccen- 
tric, and  may  be  made  more  or  less  so  by  bending  the  zuire.  The  central  dotted 
line  coincides  with  the  axis  of  the  microscope.  The  revolving  part  is  connected 
zuith  the  "  Society  Sctezu  "  by  the  small  screw  (S). 

Fig.  62.  5S,  R,and  B.  All  parts  same  as  with  Fig.  6/,  except  that  the  brush 
is  carried  by  a  sliding  cylinder  the  end  view  being  indicated  in  Fig.  63. 


64. 


65. 


66. 

Figs.  64,  65,  66.  Specimens  Showing  the  Use  of  the  Marker. 

In  Fig.  64  a  section  of  a  series  is  marked  to  indicate  that  this  section  shows  some- 
thing especially  well.  In  Fig.  65  some  blood  corpuscles  showing  ingested  carbon 
very  satisfactorily  are  surrounded  by  a  minute  ring,  and  in  Fig.  66  the  lines  of  a 
micrometer  are  ringed  to  facilitate  finding  the  lines. 


CH.  II.] 


L  A  BORA  TOR  Y  MICRO  SCOPES. 


Fig.  67.  A'rauss'  Method  of  Mark- 
ing Objectives  on  a  Revolving   Nost 
Piece. 

As  seen  in  the  figure \  the  equiva- 
lent focus  of  the  objective  is  engra ; , ,/ 
>u  the  diaphragm  above  the  back  lens 
and  may  be  very  readily  seen  in  ro- 
tating the  nose-piece.  This  is  of  great 
advantage  and  facilitates  the  chang- 
ing of  objectives,  as  one  can  see  '.chat 
objective  is  coming  into  place  with- 
out trouble. 


Figs.  68-69.     The   Tolles-Mayall  Mechanical  Stage  {\  116). 

Hath  these  mechanical  stages  have  the  great  advantage  of  large  movement  in  both 
directions,  so  that  a  series  may  be  studied  with  great  certainty  and  facility.  Both  hath 
scales  and  verniers,  so  that  the  position  of  any  particular  feature  of  a  preparation  may 
be  readily  re  found  The  figures  on  the  scale  being  different  there  is  never  doubt  as  to 
the  position  of  each  from  the  record. 

FlG.  68.    The  Tolles  Mayall  mechanical  stage  as  constructed  by  Leitz.     It  is  shown  in 
position  on  the  stage  of  the  microscope  :  it  is  fastened  to  the  stage  by  a  pin  and  s. 
near  the  pillar. 

Fig.  69.    The  Tolles  Mayall  mechanical  stage  made  by  the  Bausch  &    Lomb  Optical 
Company.     It  is  separated  from  the  microscope.     It  is  attached  to  the  microscope  by  a 
(lamp  surrounding  the  pillar.      This  form  of  connection  was  employed  by  Reicheri 
Zeiss  in  the  earlier  forms  devised,  and  is  still  used  by  them 


66 


LA  BORA  TOR  Y  MICROSCOPES. 


[CH.  II. 


Fig.  70.  Zeiss'  Microscope  1*  with  Mechanical  Stage.  This  figure,  from  Zeiss' 
Catalog  No.  30,  represents  the  Continental  Model  of  Microscope  in  its  most  per- 
fect form. 

m 

K.  Milled  head  of  the  screw  for  the  lateral  movements  of  the  stage. 

L.  Screw  for  fixing  the  laterally  moving  mechanism  of  the  stage.     By  unscrew- 
ing this  the  laterally  movi?ig  part  may  be  removed,  leaving  a  plain  stage. 

IV.  Screw  for  moving  the  stage  forward  and  backivard. 


/  ■//.  II. 


i. .  i in >a\  i  n >A')-  .ui< 'R( >sori:s. 


65  a 


Fig.  70(7 . — Spencer-Winkel  form  of  Mechanical  Stage.  It  is  readily  attachable 
to  any  microscope  with  rectangular  stage,  by  /:<.■<>  clamping  screws  on  the  right. 
Its  range  if  motion  is  about  jo  X  60  mm.,  thus  enabling  <>//<■  to  study  as  large  a 
series  as  is  usually  put  on  an  ordinary  slide. 


Fig.  ~o/>. — [See  next  page.)  Spencer  Lens  Company's  Microscope  Xo.  /.  This 
microscope  is  also  furnished  with  a  tube  ami  oculars  of  t/ic  sunn-  diameter  as  those 
on  the  Zeiss  microscopes  for  investigators  who  prefer  the  smalt  tit 


CI  I.  II] 


LA  BORA  TOR  V  MICROSCOPES. 


67 


-.::'■'■  '■' 


Fig.  71.  Watson  &  Sons'  Edinburgh  Students'  Microscope  (Stand  G).  This  is 
a  good  representative  of  the  tripod-base,  English  models  of  to-day.  It  is  in  gen- 
eral like  the  Powell  and  Lealand  stands  which  have  held  their  position  with  the 
foremost  English  microscopists  for  the  last  40  years.     (See  Carpenter-Dallinger, 

p.  172). 

Microscopes  with  tripod  bases  something  after  this  pattern  are  now  being  made 
on  the  Continent. 

Attention  is  called  to  the  sub- stage  for  the  condenser.  It  possesses  centering 
screws  so  that  any  apparatus  used  in  it  may  be  accurately  centered.  It  is  to  be 
hoped  that  all  microscopes  of  this  grade  will  soon  be  supplied  with  a  centering  sub- 
stage. 


68 


LABORATORY  MICROSCOPES. 


[CH.  II 


Fig.  72.  Natchet  &  Fils"  Medium  Microscope  No.  4  with  Movable  Stage.  The 
French  microscopes  set  the  fashion  for  the  Continent  of  Europe,  and  the  Conti- 
nental or  Harttiack  Model  with  the  horse  shoe  base  has  extended  to  all  lands,  but 
see  Carpenter ■  Dallinger ,  p.  20S. 


LA  BORA  TOR  Y  MICROSCOPES 


Fig.  73.    The  BB  Microscope  of  the  Bausch  &  Lomb  Optical  Co. 


7o 


LA  BORA  TORY  MICROSCOPES. 


\CH.  II. 


Fig.  74.  ReicherVs  Stand  III  B,  after  Specifications  by  Richards  &  Co.>  N.  Y. 


(■//.  //.] 


/. .  /  BORA  TOR  } '  MICROSCOPES. 


7' 


1  !<;   75-   Queen  &  Company's  Microscope  of  the  Continental  Pattern,   No.   II. 


72 


LABOR  A  TOR  Y  MICROSCOPES. 


\_CH.  II. 


Fig.  76.   Leitzy  Microscope,  lb. 


CI  I.   //.] 


LA  BORA  TOR  Y  MICROSCOPl.S. 


73 


Fig.  77.   Ross  Eclipse  Microscope.     The  heavy  circular  base  rotates  to g\ 

firmness  upon  inclination. 


74 


LABORATORY  MICROSCOPES. 


[CH.  II. 


Fig.  78.  The  A  A  Microscope  of  the  Bausch  &  Lomb  Optical  Co.,  with  slid- 
ing tube  instead  0/  rack  and  pinion.  This  microscope  is  also  furnished  with  rack 
and  pinion. 


CH.   //.] 


LA  BORA  TORY  MIL  ROSCOPES. 


75 


Fig.  79.    Beck's  Star  Microscope. 


76 


LABORATORY  MICROSCOPES. 


[CH.  II. 


Fig.  So.  Zeulmayer's  Clinical  Microscope.  This  is  supplied  with  a  lamp  and 
so  mounted  that  it  may  readily  be  passed  around  a  class  for  the  purpose  of  demon- 
strating some  microscopic  structure. 


Fig.  8i.  Zentmayer" s  Microscope,  No.    V. 


CII.  II] 


1. .  I  BORA  TOR ) '  .  UfCROSCOr/CS. 


77 


FlG.  82.  Leitz'  Demonstration  Microscope.  7 his  is  designed  for  class  demon- 
stration and  is  pointed  toward  the  ivindow,  or  some  other  source  of  light,  by  the 
student.  It  has  an  arrangement  for  holding  a  sketch  of  the  object  under  the 
microscope. 


Fig.  S3.  Leitz1  Microscope, 
No.  IV,  with  sliding  tube  for 
coarse  adjustment,  and  no  joint 
for  inclination.  This  micro- 
scope must  always  be  used  in  the 
vertical  position.  Microscopes 
of  this  form  are  not  so  much  the 
fashion  as  they  were. a  few  years 
ago.  If  any  instrument  re- 
quires mechanical  aids  to  en- 
able one  to  attain  a  desired  re- 
sult with  ease  and  certainty,  it 
is  a  microscope. 

Most  of  the  Leitz  microscop  - 
arc  tunc  supplied  with  joint  and 
with  rack  and  pinion  for  the 
coarse  adjustment  \  Fig,  -(>). 


78 


LA  BORA  TOR  Y  MICROSCOPES. 


[CH.  II 


Fig.  84.    Queen  <2f  Company's  Acme  Microscope ,  No.  <f> 


CH.  //.] 


LABOR.  I  TORY  M/CROSCOPES. 


79 


Fig.  85.  Mcintosh  Scientific  Microscope,  No.  2. 


CHAPTER  III. 


INTERPRETATION  OF  APPEARANCES. 


APPARATUS    AND    MATERIAL   FOR    CHAPTER    III. 

A  laboratory,  compound  microscope  (§  114)  ;  Preparation  of  fly's  wing  ;  50  per 
cent,  glycerin  ;  Slides  and  covers  ;  Preparation  of  letters  in  stairs  (Fig.  86)  ;  Muci- 
lage for  air-bubbles  and  olive  or  clove  oil  for  oil-globules  (§  127-130).  Solid  glass 
rod,  and  glass  tube  (J  135-137)  ;  Collodion  (|  137)  ;  Carmine,  India  ink,  or  lamp 
black  {I  139-141 )  ;  Frog,  castor  oil  and  micro-polariscope  (g  143). 

INTERPRETATION   OF   APPEARANCES  UNDER   THE   MICROSCOPE. 

§  120.  General  Remarks. — The  experiments  in  this  chapter  are 
given  secondarily  for  drill  in  manipulation,  but  primarily  so  that  the 
student  may  not  be  led  into  error  or  puzzled  by  appearances  which  are 
constantly  met  with  in  microscopical  investigation.  Any  one  can  look 
into  a  microscope,  but  it  is  quite  another  matter  to  interpret  correctly 
the  meaning  of  the  appearances  seen. 

It  is  especially  important  to  remember  that  the  more  of  the  relations 
of  any  object  are  known,  the  truer  is  the  comprehension  of  the  object. 
In  microscopical  investigation  every  object  should  be  scrutinized  from 
all  sides  and  under  all  conditions  in  which  it  is  likely  to  occur  in  nature 
and  in  microscopical  investigation.  It  is  best  also  to  begin  with  objects 
of  considerable  size  whose  character  is  well  known,  to  look  at  them 
carefully  with  the  unaided  eye  so  as  to  see  them  as  wholes  and  in  their 
natural  setting.  Then  a  low  power  is  used,  and  so  on  step  by  step  until 
the  highest  power  available  has  been  employed.  One  will  in  this  way 
see  less  and  less  of  the  object  as  a  whole,  but  every  increase  in  magnifi- 
cation will  give  increased  prominence  to  detail,  detail  which  might  be 
meaningless  when  taken  alone  and  independent  of  the  object  as  a  whole. 
The  pertinence  of  this  advice  will  be  appreciated  when  the  student  un- 
dertakes to  solve  the  problems  of  histology  ;  for  even  after  all  the  years 
of  incessant  labor  spent  in  trying  to  make  out  the  structure  of  man  and 
the  lower  animals,  many  details  are  still  in  doubt,  the  same  visual  ap- 
pearances being  quite  differently  interpreted  by  eminent  observers. 

Appearances  which  seem  perfectly  unmistakable  with  a  low  power 
may  be  found  erroneous  or  very  inadequate,  for  details  of  structure  that 
were  indistinguishable  with  the  low  power  may  become  perfectly  evi- 


t  ir   III  ]  TNTERPRE  TA  Tit  >N  OF  .  1 /'/'/■:.  USANCES.  8 1 

dent  with  a  higher  power  <>r  a  more  perfect  objective.     Indeed  the  prob- 
lems of  microscopic  structure  appear  to  become  ever  more  complex,  for 
difficulties  overcome  by  improvements  in  the  microscope  simply  give 
place  to  new  difficulties,  which  in  some  casts  render  the  subject  mi 
obscure  than  it  appeared  to  be  with  the  less  perfect  appliances. 

The  need  of  the  most  careful  observation  and  constant  watchfulness 
lest  the  appearances  may  be  deceptive  are  thus  admirably  stated  by  Dai- 
linger  (See  Carpenter- Dallinger,  pp.  368-369)  :  "The  correctness  of 
the  conclusions  which  the  microscopist  will  draw  regarding  the  nature 
of  any  object  from  the  visual  appearances  which  it  presents  to  him  when 
examined  in  the  various  modes  now  specified  will  necessarily  depend  in 
a  great  degree  upon  his  previous  experience  in  microscopic  observation 
and  upon  his  knowledge  of  the  class  of  bodies  to  which  the  particular 
specimen  may  belong.  Not  only  are  observations  of  any  kind  liable  to 
certain  fallacies  arising  out  of  the  previous  notions  which  the  observer 
may  entertain  in  regard  to  the  constitution  of  the  objects  or  the  nature 
of  the  actions  to  which  his  attention  is  directed,  but  even  the  most  prac- 
ticed observer  is  apt  to  take  no  note  of  such  phenomena  as  his  mind  is 
not  prepared  to  appreciate.  Errors  and  imperfections  of  this  kind  can 
only  be  corrected,  it  is  obvious,  by  general  advance  in  scientific  knowl- 
edge ;  but  the  history  of  them  affords  a  useful  warning  against  hasty 
conclusions  drawn  from  a  too  cursory  examination.  If  the  history  of 
almost  any  scientific  investigation  were  fully  made  known  it  would  gen- 
erally appear  that  the  stability  and  completeness  of  the  conclusions  fin- 
ally arrived  at  had  been  only  attained  after  many  modifications,  or  even 
entire  alterations,  of  doctrine.  And  it  is  therefore  of  such  great  impor- 
tance as  to  be  almost  essential  to  the  correctness  of  our  conclusions  that 
they  should  not  be  finally  formed  and  announced  until  they  have  been 
tested  in  every  conceivable  mode.  It  is  due  to  science  that  it  should  be 
burdened  with  as  few  false  facts  [artifacts]  and  false  doctrines  as  possi- 
ble. It  is  due  to  other  truth-seekers  that  they  should  not  be  misled,  to 
the  great  waste  of  their  time  and  pains,  by  our  errors.  And  it  is  due 
to  ourselves  that  we  should  not  commit  our  reputation  to  the  chance  of 
impairment  by  the  premature  formation  and  publication  of  conclusions 
which  may  be  at  once  reversed  by  other  observers  better  informed  than 
ourselves,  or  may  be  proved  fallacious  at  some  future  time,  perhaps  even 
by  our  own  more  extended  and  careful  researches.  The  suspension  of 
the  judgment  whenever  there  seems  room  for  doubt  is  a  lesson  inculcated 
by  all  those  philosophers  who  have  gained  the  highest  repute  for  prac- 
tical wisdom  ;  and  it  is  one  which  the  microscopist  cannot  too  soon  learn 
or  too  constantly  practice." 
6 


82  INTERPRETATION  OF  APPEARANCES.  [CH.  III. 

For  these  experiments  no  condenser  is  to  be  used  except  where  specifi- 
cally indicated. 

§  121.  Dust  or  Cloudiness  on  the  Ocular. — Employ  the  16  mm. 
(2/i  in.)  objective,  low  ocular,  and  fly's  wing  as  object. 

Unscrew  the  field-lens  and  put  some  particles  of  lint  from  dark  cloth 
on  its  upper  surface.  Replace  the  field-lens  and  put  the  ocular  in  posi- 
tion (§  44).  Eight  the  field  well  and  focus  sharply.  The  image  will 
be  clear,  but  part  of  the  field  will  be  obscured  by  the  irregular  outline 
of  the  particles  of  lint.  Move  the  object  to  make  sure  this  appearance 
is  not  due  to  it. 

Grasp  the  ocular  by  the  milled  ring,  just  above  the  tube  of  the  micro- 
scope, and  rotate  it.  The  irregular  object  will  rotate  with  the  ocular. 
Cloudiness  or  particles  of  dust  on  any  part  of  the  ocular  may  be  detect- 
ed in  this  wa5T. 

§  122.  Dust  or  Cloudiness  on  the  Objective. — Employ  the  same 
ocular  and  objective  as  before  and  the  fly's  wing  as  object.  Focus 
and  light  well,  and  observe  carefully  the  appearance.  Rub  glycerin  on 
one  side  of  a  slide  near  the  end.  Hold  the  clean  side  of  this  end  close 
against  the  objective.  The  image  will  be  obscured,  and  cannot  be  made 
clear  by  focusing.  Then  use  a  clean  slide,  and  the  image  may  be  made 
clear  by  elevating  the  tube  slightly.  The  obscurity  produced  in  this 
way  is  like  that  caused  by  clouding  the  front-lens  of  the  objective. 
Dust  would  make  a  dark  patch  on  the  image  that  would  remain  station- 
ary while  the  object  or  ocular  is  moved. 

If  a  small  diaphragm  is  employed  and  it  is  close  to  the  object,  only 
the  central  part  of  the  field  will  be  illuminated,  and  around  the  small 
light  circle  will  be  seen  a  dark  ring  (Fig.  42).  If  the  diaphragm  is 
lowered  or  a  sufficiently  large  one  employed  the  entire  field  will  be 
lighted. 

§  123.  Relative  Position  of  Objects  or  parts  of  the  same  object:. 
The  general  rule  is  that  objects  highest  up  come  into  focus  last  in 
focusing  up,  first  in  focusing  down. 

Fig.  86.  Letters  mounted  in  stairs  to  show 
the  order  of  coming  into  focus. 

a,  0,  c,  d.     The  various  letters  indicated 
by  the  oblique  rozc  of  black  marks  in  the 
sectional  vieiv. '  Slide.    The  glass  slide  on  which  the  letters  are  mounted. 

§  124.  Objects  having  Plane  or  Irregular  Outlines. — As  object 
use  three  printed  letters  in  stairs  mounted  in  Canada  balsam  (Fig.  86). 
The  first  letter  is  placed  directly  upon  the  slide,  and  covered  with  a 


a 
,b 

c 
d 

<4    1    ,   A     F.. 

CH.  11/.}  INTERPRETATION  OF  APPEARANCES.  83 

small  piece  of  glass  about  as  thick  as  a  slide.     The  second   letter   is 
placed  upon  this  and  covered  in  like  manner.     The  third  letter  is  placed 
upon  the  second  thick  cover  and  covered  with  an  ordinary  cover-gla^- 
The  letters  should  be  as  near  together  as  possible,  but  not  over-lapping. 
Employ  the  same  ocular  and  objective  as  above  ($  121). 

Lower  the  tube  till  the  objective  almost  touches  the  top  letter,  then 
look  into  the  microscope,  and  slowly  focus  up.  The  lowest  letter  will 
first  appear,  and  then,  as  it  disappears,  the  middle  one  will  appear,  and 
so  on.  Focus  down,  and  the  top  letter  will  first  appear,  then  the  mid- 
dle one,  etc.  The  relative  position  of  objects  is  determined  exactly  in 
this  way  in  practical  work. 

For  example,  if  one  has  a  micrometer  ruled  on  a  cover-glass  15-25 
hundredths  mm.  thick,  it  is  not  easy  to  determine  with  the  naked  eye 
which  is  the  ruled  surface.  But  if  one  puts  the  micrometer  under  a 
microscope  and  uses  a  3  mm.  (Jsth  in.)  objective,  it  is  easily  deter- 
mined. The  cover  should  be  laid  on  a  slide  and  focused  till  the  lines 
are  sharp.  Now,  without  changing  the  focus  in  the  least,  turn  the 
cover  over.  If  it  is  necessary  to  focus  up  to  get  the  lines  of  the  microm- 
eter sharp,  the  lines  are  on  the  upper  side.  If  one  must  focus  down, 
the  lines  are  on  the  under  surface.  With  a  thin  cover  and  delicate 
lines  this  method  of  determining  the  position  of  the  rulings  is  of  con- 
siderable practical  importance. 

ij  125.  Determination  of  the  Form  of  Objects. — The  procedure 
is  exactly  as  for  the  determination  of  the  form  of  large  objects.  That 
is,  one  must  examine  the  various  aspects.  For  example,  if  one  were 
placed  in  front  of  a  wall  of  some  kind  he  could  not  tell  whether  it  was  a 
simple  wall  or  whether  it  was  one  side  of  a  building  unless  in  some  way 
he  could  see  more  than  the  face  of  the  wall.  In  other  words,  in  order 
to  get  a  correct  notion  of  any  body,  one  must  examine  more  than  one 
dimension, — two  for  plane  surfaces,  three  for  solids.  So  for  micro- 
scopic objects,  one  must  in  some  way  examine  more  than  one  face.  To 
do  this  with  small  bodies  in  a  liquid  the  bodies  may  be  made  to  roll  over 
by  pressing  on  one  edge  of  the  cover-glass.  And  in  rolling  over  the 
various  aspects  are  presented  to  the  observer.  With  solid  bodies,  like 
the  various  organs,  correct  notions  of  the  form  of  the  elements  can  be 
determined  by  studying  sections  cut  at  right  angles  to  each  other.  The 
methods  of  getting  the  elements  to  roll  over,  and  of  sectioning  in  different 
planes  are  in  constant  use  in  histology,  and  the  microscopist  who  neglects 
to  see  all  sides  of  the  tissue  elements  has  a  very  inadequate  and  often  a 
very  erroneous  conception  of  their  true  form. 

§  126.   Transparent  Objects  having  Curved  Outlines. — The  sue- 


84  INTERPRETATION  OF  APPEARANCES.  [CH.  Ill 

cess  of  these  experiments  will  depend  entirely  upon  the  care  and  skill 
used  in  preparing  the  objects,  in  lighting,  and  in  focusing. 

Employ  a  3  mm.  (y&  in.)  or  higher  objective  and  a  high  ocular  for  all 
the  experiments.  It  may  be  necessary  to  shade  the  object  (§  102)  to 
get  satisfactory  results.  When  a  diaphragm  is  used  the  opening  should 
be  small  and  it  should  be  close  to  the  object. 

§  127.  Air  Bubbles. — Prepare  these  by  placing  a  drop  of  thin  muci- 
lage on  the  center  of  a  slide  and  beating  it  with  a  scalpel  blade  until  the 
mucilage  looks  milky  from  the  inclusion  of  air  bubbles.  Put  on  a  cover- 
glass,  but  do  not  press  it  down. 


Fig.  87.  Diagram  shozving 
how  io  place  a  cover-glass  upon 
an  object  with  fine  forceps. 


§  128.  Air  Bubbles  with  Central  Illumination. — Shade  the  ob- 
ject ;  and  with  the  plane  mirror,  light  the  field  with  central  light  (Fig. 

23)- 

Search  the  preparation  until  an  air  bubble  is  found  appearing  about 
1  mm.  in  diameter,  get  it  into  the  center  of  the  field,  and  if  the  light  is 
central  the  air  bubble  will  appear  with  a  wide,  dark,  circular  margin 
and  a  small  bright  center.  If  the  bright  spot  is  not  in  the  center,  ad- 
just the  mirror  until  it  is. 

This  is  one  of  the  simplest  and  surest  methods  of  telling  when  the 
light  is  central  or  axial  when  no  condenser  is  used  (§  61). 

Focus  both  up  and  down,  noting  that,  in  focusing  up,  the  central  spot 
becomes  very  clear  and  the  black  ring  very  sharp.  On  elevating  the 
tube  of  the  microscope  still  more  the  center  becomes  dim,  and  the  whole 
bubble  loses  its  sharpness  of  outline. 

§  129.  Air  Bubbles  with  Oblique  Illumination.— Remove  the  sub- 
stage  of  the  microscope  and  all  the  diaphragms.  Swing  the  mirror  so 
that  the  rays  may  be  sent  very  obliquely  upon  the  object  (Fig.  23,  C). 
The  bright  spot  will  appear  no  longer  in  the  center  but  on  the  side  away 
from  the  mirror  (Fig.  88). 

§  130.  Oil  Globules. — Prepare  these  by  beating  a  small  drop  of  clove 
oil  with  mucilage  on  a  slide  and  covering  as  directed  for  air  bubbles 
(§  128),  or  use  a  drop  of  milk. 

§  131.  Oil  Globules  with  Central  Illumination.  — Use  the  same 
diaphragm  and  light  as  above  (§  128).  Find  an  oil  globule  appearing 
about  1  mm.  in  diameter.     If  the  light  is  central  the  bright  spot  will  ap- 


CH.  Ill]  INTERPRETATION  OF  APPEARANCES.  85 

pear  in  the  center  as  with  air.     Focus  up  and  down  as  with  air,  and  note 
that  the  bright  center  of  the  oil  globule  is  clearest  last  in  focusing  up. 

FlG.  88.     Very  small  Globule  of  Oil  (O)  and  an  Air  Hubble  (A)  a 

seen  by  Oblique  Light.     The  arrow  indicates  the  direction  of  the 
light  rays. 

£  132.  Oil  Globules  with  Oblique  Illumination. — Re- 
move the  sub-stage,  etc.,  as  above,  and  swing  the  mirror  to 
one  side  and  light  with  oblique  light.  The  bright  spot  will 
be  eccentric,  and  will  appear  to  be  on  the  same  side  as  the 
mirror  (Fig.  88). 

§  l33-  Oil  and  Air  Together. — Make  a  preparation  ex- 
actly as  described  for  air  bubbles  (§  127),  and  add  at  one 
edge  a  little  of  the  mixture  of  oil  and  mucilage  (§  130)  ; 
cover  and  examine. 

The  sub-stage  need  not  be  used  in  this  experiment.  Search  the  prep- 
aration until  an  air  bubble  and  an  oil  globule,  each  appearing  about  1 
mm.  in  diameter,  are  found  in  the  same  field  of  view.  Light  first  with 
central  light,  and  note  that,  in  focusing  up,  the  air  bubble  comes  into 
focus  first  and  that  the  central  spot  is  smaller  than  that  of  the  oil  globule. 
Then,  of  course,  the  black  ring  will  be  wider  in  the  air  bubble  than  in 
the  oil  globule.  Make  the  light  oblique.  The  bright  spot  in  the  air 
bubble  will  move  away  from  the  mirror  while  that  in  the  oil  globule  will 
move  toward  it.     See  Fig.  88.* 

§  134.  Air  and  Oil  by  Reflected  Light. — Cover  the  diaphragm  or 
mirror  so  that  no  transmitted  light  f§  60)  can  reach  the  preparation, 
using  the  same  preparation  as  in  §  133.  The  oil  and  air  will  appear 
like  globules  of  silver  on  a  dark  ground.  The  part  that  was  darkest  in 
each  will  be  lightest,  and  the  bright  central  spot  will  be  somewhat 
dark.t 

£  135.   Distinctness  of  Outline. — In  refraction  images  this  depends 

on  the  difference  between  the  refractive  power  of  a  body  and  that  of  the 
medium  which  surrounds  it.      The  oil  and  air  were  very  distinct  in  out- 

*  It  should  be  remembered  that  the  image  in  the  compound  microscope  is  in- 
verted (Fig.  2i ),  hence  the  bright  spot  really  moves  toward  the  mirror  for  air,  and 
away  from  it  for  oil. 

f  It  is  possible  to  distinguish  oil  and  air  optically,  as  described  above,  only  when 
quite  high  powers  are  used  and  very  small  bubbles  are  selected  for  observation.  If 
a  16  mm.  (^in.)is  used  instead  of  a  3  mm.  ('sin.)  objective,  the  appearances 
will  vary  considerably  from  that  given  above  for  the  higher  power.  It  is  well  to 
use  a  low  as  well  as  a  high  power.  Marked  differences  will  also  be  seen  in  the  ap- 
pearances with  objectives  of  small  and  of  large  aperture. 


36  INTERPRETATION  OF  APPEARANCES.  \_CH.  III. 

line  as  both  differ  greatly  in  refractive  power  from  the  medium  which 
surrounds  them,  the  oil  being  more  refractive  than  the  mucilage  and 
the  air  less.      (Figs.  53-55) • 

Place  a  fragment  of  a  cover-glass  on  a  clean  slide,  and  cover  it  (see 
under  mounting).  The  outline  will  be  very  distinct  with  the  unaided 
eye.  Use  it  as  object  and  employ  the  16  mm.  (73  in.)  objective  and 
high  ocular.  Light  with  central  light.  The  fragment  will  be  outlined 
by  a  dark  band.  Put  a  drop  of  water  at  the  edge  of  the  cover-glass. 
It  will  run  in  and  immerse  the  fragment.  The  outline  will  remain  dis- 
tinct, but  the  dark  band  will  be  somewhat  narrower.  Remove  the  cover- 
glass,  wipe  it  dry,  and  wipe  the  fragment  and  slide  dry  also.  Put  a 
drop  of  50%  glycerin  on  the  middle  of  the  slide  and  mount  the  fragment 
of  cover-glass  in  that.  The  dark  contour  will  be  much  narrower  than 
before. 

Draw  a  solid  glass  rod  out  to  a  fine  thread.  Mount  one  piece  in  air, 
and  the  other  in  50^  glycerin.  Put  a  cover-glass  on  each.  Employ 
the  same  optical  arrangement  as  before.  Examine  the  one  in  air  first. 
There  will  be  seen  a  narrow,  bright  baud,  with  a  wide,  dark  baud  on 
each  side. 

The  one  in  glycerin  will  show  a  much  wider  bright  central  band,  with 
the  dark  borders  correspondingly  narrow  ( Fig.  89  b).  The  dark  contour 
depends  also  on  the  numerical  aperture  of  the  objective — being  wider 
with  low  apertures.  This  can  be  readily  understood  when  it  is  remem- 
bered that  the  greater  the  aperture  the  more  oblique  the  rays  of  light 
that  can  be  received,  and  the  dark  band  simply  represents  an  area  in 
which  the  rays  are  so  greatly  bent  or  refracted  (Figs.  53,  55)  that  they 
cannot  enter  the  objective  and  contribute  to  the  formation  of  the  image  ; 
the  edges  are  dark  simply  because  no  light  from  them  reaches  the  ob- 
server. 

Fig.  89.   Solid  glass  rod  showing  the  ap- 
pearance  when   viewed  with   transmitted, 
central  light,  and  with  an  objective  of  medi- 
um aperture. 
a.  Mounted  in  air.     b.  Mounted  in  50  per  cent,  glycerin. 

If  the  glass  rod  or  any  other  object  were  mounted  in  a  medium  of  the 
same  color  and  refractive  power,  it  could  not  be  distinguished  from  the 
medium.* 

*  Some  of  the  rods  have  air  bubbles  in  them,  and  then  there  results  a  capillary 
tube  when  they  are  drawn  out.  It  is  well  to  draw  out  a  glass  tube  into  a  fine  thread 
and  examine  it  as  described.  The  central  cavity  makes  the  experiment  much  more 
complex. 


CH.  IIL}  INTERPRETATION  OF  APPEARANCES.  87 

A  very  striking  and  satisfactory  demonstration  may  be  made  by  paint- 
ing a  zone  or  band  of  eosin  or  otber  transparent  color  on  a  solid  gla- 
red, and  immersing  the  rod  in  a  test  tube  or  vial  of  cedar  oil,  clove  oil 
or  turpentine.  Above  the  liquid  the  glass  rod  is  very  evident,  as  it  is 
also  at  the  colored  zone,  but  at  other  levels  it  can  hardly  be  seen  in  the 
liquid. 

§  136.  Highly  Refractive. — This  expression  is  often  used  in  de- 
scribing microscopic  objects,  (medulated  nerve  fibers,  for  example), 
and  means  that  the  object  will  appear  to  be  bordered  by  a  wide,  dark 
margin  when  it  is  viewed  by  transmitted  light.  And  from  the  above 
(§  !35)>  ^  would  be  known  that  the  refractive  power  of  the  object,  and 
the  medium  in  which  it  was  mounted  must  differ  considerably. 

§  137.  Doubly  Contoured. — This  means  that  the  object  is  bounded 
by  two,  usually  parallel  dark  lines  with  a  lighter  band  between  them. 
In  other  words,  the  object  is  bordered  by  (1)  a  dark  line,  (2)  a  light 
band,  and  (3)  a  second  dark  line  (Fig.  90). 

This  may  be  demonstrated  by  coating  a  fine  glass  rod  (§  135)  with 
one  or  more  coats  of  collodion  or  celloidin  and  allowing  it  to  dry,  and 
then  mounting  in  50%  glycerin  as  above.  Employ  a  3  mm.  (}i  in.  )  or 
higher  objective,  light  with  transmitted  light,  and  it  will  be  seen  that 
where  the  glycerin  touches  the  collodion  coating  there  is  a  dark  line — 
next  this  is  a  light  baud,  and  finally  there  is  a  second  dark  line  where 
the  collodion  is  in  contact  with  the 
glass  rod.*     (Fig.  90). 

Fig.  90.  Solid  glass  rod  coaled  with  col- 
lodion to  show  a  double  contour.  Toward 
one  end  the  collodion  had  gathered  in  a  fusi- 
form drop. 

§  138.  Optical  Section. — This  is  the  appearance  obtained  in  examin- 
ing transparent  or  nearly  transparent  objects  with  a  microscope  when 
some  plane  below  the  upper  surface  of  the  object  is  in  focus.  The  upper 
part  of  the  object  which  is  out  of  focus  obscures  the  image  but  slightly. 
By  changing  the  position  of  the  objective  or  object,  a  different  plane  will 
be  in  focus  and  a  different  optical  section  obtained.  The  most  satisfac- 
tory optical  sections  are  obtained  with  high  objectives  having  large 
aperture. 

Nearly  all  the  transparent  objects  studied  may  be  viewed  in  optical 


*  The  collodion  used  is  a  6%  solution  of  gun  cotton  in  equal  parts  of  sulphuric 
ether  and  95%  alcohol.  It  is  well  to  dip  the  rod  two  or  three  times  in  the  collo- 
dion and  to  hold  it  vertically  while  drying.  The  collodion  will  gather  in  drops, 
and  one  will  see  the  difference  between  a  thick  and  a  thin  membranous  covering. 
(Fig.  90). 


88  INTERPRETATION  OF  APPEARANCES.  [CH.  III. 

section.  A  striking  example  will  be  found  in  studying  mammalian  red 
blood-copuscles  on  edge.  The  experiments  with  the  solid  glass  rods 
(Fig.  89)  furnish  excellent  and  striking  examples  of  optical  sections. 

§  139.  Currents  in  Liquids. — Employ  the  16  mm.  (7/3  in.)  object- 
ive, and  as  object  put  a  few  particles  of  carmine  on  the  middle  of  a  slide, 
and  add  a  drop  of  water.  Grind  the  carmine  well  with  a  scalpel  blade, 
and  then  cover  it.  If  the  microscope  is  inclined,  a  current  will  be  pro- 
duced in  the  water,  and  the  particles  of  carmine  will  be  carried  along 
by  it.  Note  that  the  particles  seem  to  flow  up  instead  of  down—why 
is  this  ? 

Lamp-black  rubbed  in  water  containing  a  little  mucilage  answers  well 
for  this  experiment. 

§  140.   Velocity  Under  the  Microscope. — In  studying  currents  or 
the  movement  of  living  things  under  the  microscope,  one  should  not 
forget  that  the  apparent  velocity  is  as  unlike  the  real  velocity  as  the  ap- 
parent size  is  unlike  the  real  size.      If  one  consults  Fig.  37  it  will  be 
seen  that  the  actual  size  of  the  field  of  the  microscope  with  the  different 
objectives  and  oculars  is  inversely  as  the  magnification.     That  is,  with 
great  magnification  only  a  small  area  can  be  seen.     The  field  appears  to 
be  large,  however,  and  if  any  object  moves  across  the  field  it  may  ap- 
pear to  move  with  great  rapidity,  whereas  if  one  measures  the  actual 
distance  passed  and  notes  the  time,  it  will  be  seen  that  the  actual  motion 
is  quite  slow.     One  should  keep  this  in  mind  in  studying  the  circulation 
of  the  blood.     The  truth  of  what  has  just  b^en  said  can  be  easily  dem- 
onstrated in  studying  the  circulation  in  the  gills  of  Necturus,  or  in  the 
frog's  foot,  by  using  first  a  low  power  in  which  the  field  is  actually  of 
considerable  diameter   (Fig.  37,  Table,  §  47)   and  then  using  a  high 
power.     With  the  high  power  the  apparent  motion  will  appear  much 
more  rapid.     For  the  form  of  motion,  spiral,  serpentine,  etc.,  see  Car- 
penter-Dallinger,  p.  375. 

§  141.  Pedesis  or  Brownian  Movement. — Employ  the  same  ob- 
ject as  above,  but  a  3  mm.  (  }i'm. )  or  higher  objective  in  place  of  the  16 
mm.  Make  the  body  of  the  microscope  vertical,  so  that  there  may  be 
no  currents  produced.  Use  a  small  diaphragm  and  light  the  field  well. 
Focus,  and  there  will  be  seen  in  the  field  large  motionless  masses,  and 
between  them  small  masses  in  constant  motion.  This  is  an  indefinite, 
dancing  or  oscillating  motion. 

This  indefinite  but  continuous  motion  of  small  particles  in  a  liquid  is 
called  P'e-d'e'sis  or  Brownian  movement.  Also,  but  improperly,  molecu- 
lar movement,  from  the  smallness  of  the  particles. 

The  motion  is  increased  by  adding  a  little  gum  arabic  solution  or  a 


CI  I.  ///,]  INTERPRETATION  OF  APPEAR  A.XCES.  80 

slight  amount  of  silicate  of  soda  or  of  soap  ;  sulphuric  acid  and  various 
saline  compounds  retard  or  check  the  motion.     One  of  the  best  obje 
is  lamp-black  ground  up  with  a  little  gum  arabic.     Carmine  prepared  in 

the  same   way,   or  simply  in  water,  is  excellent  ;   and  very  finely  pow- 
dered pumice-stone  in  water  has  for  many  years  been  a  favorite  object. 

Pedesis  is  exhibited  by  all  solid  matter  it"  it  is  finely  enough  divided 
and  in  a  suitable  liquid.  In  the  minds  of  most,  no  adequate  explana- 
tion has  yet  been  offered.  See  Carpenter-Dallinger,  p.  373  ;  Beale,  p. 
195  ;  Jevons,  in  Quart.  Jour.  Science,  n.  s. ,  Vol.  VIII  (1878),  p.  167. 
In  1894  Meade  Bache  published  a  paper  in  the  Proc.  Amer.  Philos.  Soc, 
Vol.  XXXIII,  pp.  163-167,  entitled  "The  Secret  of  the  Brownian 
Movement."     This  paper  is  suggestive  if  not  wholly  satisfactory. 

For  the  original  account  of  this  see  Robert  Brown,  "  Botanical  appen- 
dix to  Captain  King's  voyage  to  Australia,"  Vol.  II,  p.  534.     (1826). 

See  also  Dr.  C.  Aug.  Sigm.  Schultze,  "  Mikroskopische  Untersuch- 
ungen  iiber  des  Herren  Robert  Brown  Entdeckung  lebender,  selbst  im 
Feuer  unzerstorbarer  Theilchen  in  alien  Korpern. ' '  From  ' '  Die  Gesell- 
schaft  fiir  Beforderung  der  Naturwissenschaften  zu  Freiburg."    1828. 

Compare  the  pedetic  motion  with  that  of  a  current  by  slightly  inclin- 
ing the  tube  of  the  microscope.  The  small  particles  will  continue  their 
independent  leaping  movements  while  they  are  carried  along  by  the 
current. 

§  142.  Demonstration  of  Pedesis  with  the  Polarizing  Micro- 
scope.— The  following  demonstration  shows  conclusively  that  the  pe- 
detic motion  is  real  and  not  illusive.      (Rauvier,  p.  173). 

Open  the  abdomen  of  a  dead  frog  (an  alcoholic  specimen  will  do  if  it 
is  soaked  in  water  for  some  time,  but  a  fresh  specimen  is  more  satisfac- 
tory). Turn  the  viscera  to  one  side  and  observe  the  small,  whitish 
masses  at  the  emergence  of  the  spinal  nerves.  With  fine  forceps  remove 
one  of  these  and  place  it  on  the  middle  of  a  clean  slide.  Add  a  drop  of 
water,  or  of  water  containing  a  little  gum  arabic.  Rub  the  white  mass 
around  in  the  drop  of  liquid  and  soon  the  liquid  will  have  a  milky  ap- 
pearance. Remove  the  white  mass,  place  a  cover-glass  on  the  milky 
liquid  and  seal  the  cover  by  painting  a  ring  of  castor  oil  all  around  it, 
half  the  ring  being  on  the  slide  and  half  on  the  cover-glass.  This  is  to 
avoid  the  production  of  currents  by  evaporation. 

Put  the  preparation  under  the  microscope  and  examine  with,  firs 
low  then  a  high  power  (3  mm.  or   ',;  in.  ).      In  the  field  will  be  seen 
multitudes  of  crystals  of  carbonate  of  lime  ;  the  larger  crystals  are  mo- 
tionless but  the  smallest  ones  exhibit  marked  pedetic  movement. 

Use  the  micro-polariscope,  light  with  great  care  and  exclude  all  ad 


90  INTERPRETATION  OF  APPEARANCES.  [CH.  III. 

ventitious  liglit  from  the  microscope  by  shading  the  object  (§  102)  and 
also  by  shading  the  eye.  Focus  sharply  and  observe  the  pedetic  motion 
of  the  small  particles,  then  cross  the  polarizer  and  analyzer,  that  is,  turn 
one  or  the  other  until  the  field  is  dark.  Part  of  the  large,  motionless 
crystals  will  shine  continuously  and  a  part  will  remain  dark,  but  the 
small  crystals  between  the  large  ones  will  shine  for  an  instant,  then  dis- 
appear, only  to  appear  again  the  next  instant.  This  demonstration  is 
believed  to  furnish  absolute  proof  that  the  pedetic  movement  is  real  and 
not  illusory. 

§  143.  Muscae  Volitantes. — These  specks  or  filaments  in  the  eyes 
due  to  minute  shreds  or  opacities  of  the  vitreous  sometimes  appear  as  part 
of  the  object  as  they  are  projected  into  the  field  of  vision.  They  may 
be  seen  by  looking  into  the  well  lighted  microscope  when  there  is  no  ob- 
ject under  the  microscope.  They  may  also  be  seen  by  looking  at  the 
brightly  illuminated  snow  or  other  white  surface.  By  studying  them 
carefully  it  will  be  seen  that  they  are  somewhat  movable  and  float  across 
the  field  of  vision,  and  thus  do  not  remain  in  one  position  as  do  the  ob- 
jects under  observation.  Furthermore,  one  may,  by  taking  a  little 
pains,  familiarize  himself  with  the  special  forms  in  his  own  eyes  so  that 
the  more  conspicuous,  at  least,  may  be  instantly  recognized. 

§  144.  In  addition  to  the  above  experiments  it  is  very  strongly  rec- 
ommended that  the  student  follow  the  advice  of  Beale,  p.  248,  and  ex- 
amine first  with  a  low  then  a  higher  power,  mounted  dry,  then  in  water, 
lighted  with  reflected  light,  then  with  transmitted  light,  the  following  : 
Potato,  wheat,  rice,  and  corn  starch,  easily  obtained  by  scraping  the 
potato  and  the  grains  mentioned  ;  bread  crumbs  ;  portions  of  feather. 
Portions  of  feather  accidentally  present  in  histological  preparations  have 
been  mistaken  for  lymphatic  vessels  (Beale,  288).  Fibers  of  cotton, 
linen  and  silk.  Textile  fibres  accidentally  present  have  been  considered 
nerve  fibres,  etc.  Human  and  animal  hairs.  Study  with  especial  care 
hairs  from  various  parts  of  the  body  of  the  animals  used  for  dissection 
in  the  laboratory  where  you  work.  These  are  liable  to  be  present  in 
histological  preparations,  and  unless  their  character  is  understood  there 
is  chance  for  much  confusion  and  erroneous  interpretation.  The  scales 
of  butterflies  and  moths,  especially  the  common  clothes  moth.  The 
dust  swept  from  carpeted  and  wood  floors.  Tea  leaves  and  coffee 
grounds.  Dust  found  in  living  rooms  and  places  not  frequently  dusted. 
In  the  last  will  be  found  a  regular  museum  of  objects. 

For  figures  (photo-micrographs,  etc.)  of  the  various  forms  of  starch, 
see  Bulletin  No.  13  of  the  Chemical  Division  of  the  U.  S.  Department 
of  Agriculture.     For  Hair  and  Wool,  see  Bulletin  of  the  National  Asso- 


CH.  Ill}  INTERPRETATION  OF  APPEARANCES.  91 

ciation  of  Wool  Growers,  1875,  p.  470,  Proc.  Amer.  Micr.  vSoc,  1884, 
pp.  65-68. 

For  different  appearances  due  to  the  illuminator,  see  Nelson,  in  Jour. 
Roy.  Micr.  Soc.,  1891,  pp.  90-105  ;  and  for  the  illusory  appearances  due 
to  diffraction  phenomena,  see  Carpenter-Dallinger,  p.  376. 

If  it  is  necessary  to  see  all  sides  of  an  ordinary  gross  object,  and  to 
observe  it  with  varying  illumination  and  under  various  conditions  of 
temperature,  moisture,  etc.,  in  order  to  obtain  a  fairly  accurate  and 
satisfactory  knowledge  of  it,  so  much  the  more  is  it  necessary  not  to  be 
satisfied  in  microscopical  observation  until  every  means  of  investigation 
and  verification  has  bsen  called  into  service,  and  then  of  the  image  that 
falls  upon  the  retina,  only  such  details  will  be  noted  as  the  brain  behind 
the  eye  is  ready  to  appreciate. 

To  summarize  this  chapter  and  leave  with  the  beginning  student  the 
result  of  the  experience  of  many  eminent  workers  : 

1.  Get  all  the  information  possible  with  the  unaided  eye.  See  the 
whole  object  and  all  sides  of  it,  so  far  as  possible. 

2.  Examine  the  preparation  with  a  simple  microscope  in  the  same 
thorough  way  for  additional  detail. 

3.  Use  a  low  power  of  the  compound  microscope. 

4.  Use  a  higher  power. 

5.  Use  the  highest  power  available  and  applicable.  In  this  way  one 
sees  the  object  as  a  whole  and  progressively  more  and  more  details. 
Then  as  the  object  is  viewed  from  two  or  more  aspects,  something  like 
a  correct  notion  may  be  gained  of  its  form  and  structure. 


CHAPTER  IV. 


MAGNIFICATION  AND  MICROMETRY. 


APPARATUS   AND    MATERIAL   FOR   THIS    CHAPTER. 

Simple  and  compound  microscope  ;  Steel  scale  or  rule  divided  to  millimeters  and 
iths  ;  Block  for  magnifier  and  compound  microscope  (§  147,  151)  ;  Dividers  [\  147, 
151)  ;  Stage  micrometer  {\  150)  ;  Wollaston  camera  lucida  (§  151)  ;  Ocular  screw- 
micrometer  (Fig.  100)  ;  Micrometer  ocular  (Figs.  98-99).  Abbe  camera  lucida 
(Fig.  96). 

§  145.  The  Magnification,  Amplification  or  Magnifying  Power 
of  a  simple  or  compound  microscope  is  the  ratio  between  the  real  and 
the  apparent  size  of  the  object  examined.  The  apparent  size  is  obtained 
by  measuring  the  virtual  image  (Figs.  21,  38).  The  object  for  deter- 
mining magnification  must  be  of  known  length  and  is  designated  a  mi- 
crometer (§  150).  In  practice  a  virtual  image  is  measured  by  the  aid  of 
some  form  of  camera  lucida  (Figs.  92,  96),  or  by  double  vision  (§  147). 
As  the  length  of  the  object  is  known,  the  magnification  is  easily  deter- 
mined hy  dividing  the  apparent  size  of  the  image  by  the  actual  size  of 
the  object.  For  example,  if  the  virtual  image  measures  40  mm.  and 
the  object  magnified,  2  mm.,  the  amplification  must  be  40  —  2  =  20, 
that  is,  the  apparent  size  is  20  fold  greater  than  the  real  size. 

Magnification  is  expressed  in  diameters  or  times  linear,  that  is,  but 
one  dimension  is  considered.  In  giving  the  scale  at  which  a  microscop- 
ical or  histological  drawing  is  made,  the  word  magnification  is  frequent- 
ly indicated  by  the  sign  of  multiplication  thus  :  X  450,  upon  a  drawing 
would  mean  that  the  figure  or  drawing  is  450  times  as  large  as  the  object. 

§  146.  Magnification  of  Real  Images. — In  this  case  the  magnifi- 
cation is  the  ratio  between  the  size  of  the  real  image  and  the  size  of  the 
object,  and  the  size  of  the  real  image  can  be  measured  directly.  By 
recalling  the  work  on  the  function  of  an  objective  (§49),  it  will  be 
remembered  that  it  forms  a  real  image  on  the  ground  glass  placed  on 
the  top  of  the  tube,  and  that  this  real  image  could  be  looked  at  with  the 
eye  or  measured  as  if  it  were  an  actual  object.  For  example,  suppose 
the  object  were  3  millimeters  long  and  its  image  on  the  ground  glass 
measured  15  mm.,  then  the  magnification  must  be,  15  -*-  3  =  5,  that  is, 


CH.  TV.]  MAGNIFICA  TION  AND  MICRi  WE  TR ) '.  93 

the  real  image  is  5  times  as  long  as  the  object.  The  real  images  seen 
in  photography  are  mostly  smaller  than  the  objects,  but  the  magnifica- 
tion is  designated  in  the  same  way  by  dividing  the  size  of  the  real  im- 
age measured  on  the  ground  glass  by  the  size  of  the  object.  For  ex- 
ample, if  the  object  is  400  millimeters  long  and  its  image  on  the  ground 
glass  is  25  mm.  long,  the  ratio  is  25  -k-  400  =  l1T.  That  is,  the  image 
is  Y(jth  as  long  as  the  object  and  is  not  magnified  but  reduced.  In 
marking  negatives,  as  with  drawings,  the  sign  of  multiplication  is  put 
before  the  ratio,  and  in  the  example  the  designation  would  be  x  ^th. 

MAGNIFICATION   OF    A   SIMPLE   MICROSCOPE. 

^  147.  The  Magnification  of  a  Simple  Microscope  is  the  ratio 
between  the  object  magnified  (Fig.  16,  A'B'),  and  the  virtual  image 
(A3B3).  To  obtain  the  size  of  this  virtual  image  place  the  tripod  mag- 
nifier near  the  edge  of  a  support  of  such  a  height  that  the  distance  from 
the  upper  surface  of  the  magnifier  to  the  table  is  250  millimeters. 

As  object,  place  a  scale  of  some  kind  ruled  in  millimeters  on  the  sup- 
port under  the  magnifier.  Put  some  white  paper  on  the  table  at  the 
base  of  the  support,  and  on  the  side  facing  the  light. 

Close  one  eye,  and  hold  the  head  so  that  the  other  will  be  near  the 
upper  surface  of  the  lens.  Focus  if  necessary  to  make  the  image  clear 
(§  9).  Open  the  closed  eye,  and  the  image  of  the  rule  will  appear  as 
if  on  the  paper  at  the  base  of  the  support.  Hold  the  head  very  still, 
and,  with  dividers,  get  the  distance  between  any  two  lines  of  the  image. 
This  is  the  so-called  method  of  binocular  or  double  vision  in  which  the 
microscopic  image  is  seen  with  one  e)re  and  the  dividers  with  the  other, 
the  two  images  appearing  to  be  fused  in  a  single  visual  field. 

§  148.  Measuring  the  Spread  of  Dividers. — This  should  be  done 
on  a  steel  scale  divided  to  millimeters  and  iths. 

D 

As  \  mm.  cannot  be  seen  plainly  by  the  unaided  eye,  place  one  arm 
of  the  dividers  at  a  centimeter  line,  and  then  with  the  tripod  magnifier 
count  the  number  of  spaces  on  the  rule  included  between  the  points  of 
the  dividers.  The  magnifier  simply  makes  it  easy  to  count  the  spaces 
on  the  rule  included  between  the  points  of  the  dividers — it  does  not,  of 
course,  increase  the  number  of  spaces  or  change  their  value. 

As  the  distance  between  any  two  lines  of  the  image  of  the  scale  gives 
the  size  of  the  virtual  image  (Fig.  16,  A3B3),  and  as  the  size  of  the  ob- 
ject is  known,  the  magnification  is  determined  by  dividing  the  size  of 
image  by  the  size  of  the  object.  Thus,  suppose  the  distance  between 
the  two  lines  of  the  image  is  measured  by  the  dividers  and  found  on  the 


94  MAGNIFICATION  AND  MICROMETRY.  [CH.  IV. 

steel  scale  to  be  15  millimeters,  and  the  actual  size  of  the  space  between 
the  two  lines  of  the  object  is  2  millimeters,  then  the  magnification  must 
bei5-r-2  =  7^.  That  is,  the  image  is  7^  times  as  long  or  wide  as 
the  object.  In  this  case  the  image  is  said  to  be  magnified  7^  diame- 
ters, or  7*2  times  linear. 

The  magnification  of  any  simple  magnifier  may  be  determined  exper- 
imentally in  the  way  described  for  the  tripod. 

MAGNIFICATION    OF    A    COMPOUND    MICROSCOPE. 

§  149.  The  Magnification  of  a  Compound  Microscope  is  the 
ratio  between  the  final  or  virtual  image  (Fig.  21,  B3A8),  and  the  object 
magnified  (A  B)., 

The  determination  of  the  magnification  of  a  compound  microscope  may 
be  made  as  with  a  simple  microscope  (§  147),  but  this  is  very  fatiguing 
and  unsatisfactory. 

§  150.  Stage,  Object  or  Objective  Micrometer. — For  determin- 
ing the  magnification  of  a  compound  microscope  and  for  the  purposes 
of  micrometry,  it  is  necessary  to  have  a  finely  divided  scale  or  rule  on 
glass  or  on  metal.  Such  a  finely  divided  scale  is  called  a  micrometer, 
and  for  ordinary  work  one  on  glass  is  most  convenient.  The  spaces 
between  the  lines  should  be  ^  and  T\-§  millimeter,  and  when  high  pow- 
ers are  to  be  used  the  lines  should  be  very  fine.  It  is  of  advantage  to 
have  the  coarser  lines  filled  with  graphite  (plumbago),  especially  when 

low  powers  are  to  be  used.  If  one  has 
an  uncovered  micrometer  the  lines  may 
be  very  readily  filled  by  rubbing  some  of 
the  plumbago  on  the  surface  with  the  end 
of  a  cork  ;  the  superfluous  plumbago  may 
Fig.  91.  Diagram  of  a  stage  be  removed  by  using  a  clean  dry  cloth  or 
micrometer,  with  a  ring  on  the     a  piece  of  lens  paper.      After  the  lines  are 

lines  to  facilitate  finding  them.         £11    ,         ,  ,,         11  •      j    r  ^ 

J  j         &  filled  and  the  plumbago  wiped  from  the 

surface,  the  slide  should  be  examined  and  if  it  is  found  satisfactory,  i.  e. , 

if  the  lines  are  black,  a  cover-glass  on  which  is  a  drop  of  warm  balsam 

may  be  put  over  the  lines  to  protect  them. 

If  one  desires  to  have  a  part  of  the  micrometer  uncovered  and  a  part 
covered  for  using  homogeneous  objectives,  the  lines  may  be  filled  with 
fine  graphite,  as  described,  and  a  piece  of  oblong  cover-glass  placed  over 
a  part  of  the  band  of  lines. 

§  151.  Determination  of  Magnification.  — This  is  most  readily  ac- 
complished by  the  use  of  some  form  of  camera  lucida  (Ch.  V),  that  of 


CH.  IV.] 


MAGNIFICATION  AMD  MICROMETRY. 


95 


Wollaston  being  most  convenient  as  it  may  be  used  for  all  powers, 
and  the  determination  of  the  standard  distance  of  250  millimeters  at 
which  to  measure  the  image  is  very  readily  determined  (Fig.  92,  $  153). 
Employ  the  16  mm.  (;j  in.)  objective  and  a  50mm.  (2  in.,  A.  or 
No.  1)  ocular  and  stage  micrometer  as  object.  For  this  power  the  -,\yth 
mm.  spaces  of  the  micrometer  should  be  used  as  object.  Focus  sharply, 
and   make  the  tube  of  the  microscope  horizontal,  by  bending  the  flexible 

Fig.  92.  Wollaston' s  Camera  Luci- 
da, showing  the  rays  from  the  micro- 
scope and  from  the  drazving  surface, 
and  the  position  of  the  pupil  of  the  eye. 

Axis,  Axis.  Axial  rays  from  the 
microscope  and  from  the  drawing  sur- 
face {Ch.  V). 

Camera  Lucid  a.  A  section  of  the 
quadrangular  prism  showing  the 
course  of  the  rays  in  the  pt  ism  from 
the  microscope  to  the  eye.  As  the  rays 
are  twice  refected,  they  have  the  same 
relation  on  entering  the  eye  that  they 
would  have  by  looking  directly  into  the 
ocular. 

A  B.  The  lateral  rays  from  the  mi- 
croscope and  their  projection  on  the 
drazving  surface. 

CD.  Rays  from  the  drawing  sur- 
face to  the  eye. 


Fig.  92 


A  D,  A'  D' .  Overlapping  portion  of  the  two  fields,  where  both  the  microscopic 
image  and  the  drawing  surface,  pencil,  etc.,  may  be  seen.  It  is  represented  by  the 
shaded  part  in  the  overlapping  circles  at  the  right. 

Ocular.   The  ocular  of  the  microscope. 

P.    The  drawing  pencil.     Its  point  is  shown  in  the  overlapping  fields. 

pillar,  being  careful  not  to  bring  any  strain  upon  the  fine  adjustment. 
(Frontispiece). 

Put  a  Wollaston  camera  lucida  (Ch.  V.)  in  position,  and  turn  the  oc- 
ular around  if  necessary  so  that  the  broad  fiat  surface  may  face  directly 
upward,  as  shown  in  Fig.  92.  Elevate  the  microscope  by  putting  a 
block  under  the  base,  so  that  the  perpendicular  distance  from  the  upper 
surface  of  the  camera  lucida  to  the  table  is  250  mm.  (§  153).  Place 
some  white  paper  011  the  work-table  beneath  the  camera  lucida. 

Close  one  eye,  and  hold  the  head  so  that  the  other  may  be  very  close 
to  the  camera  lucida.  Look  directly  down.  The  image  will  appear  to 
be  on  the  table.  It  may  be  necessary  to  readjust  the  focus  after  the 
camera  lucida  is  in  position.      If  there  is  difficulty  in  seeing  dividers  and 


96 


MAGNIFICATION  AND  MICROMETRY. 


\CH.  IV. 


image  consult  Ch.  V.     Measure  the  image  with  dividers  and  obtain  the 
power  exactly  as  above  (§  147,  148). 

Thus  :  Suppose  two  of  the  yVth  mm.  spaces  were  taken  as  object,  and 
the  image  is  measured  by  the  dividers,  and  the  spread  of  the  dividers  is 
found  on  the  steel  rule  to  be  gf  millimeters.  If  now  the  object  is  y-yths 
of  a  millimeter  and  the  magnified  image  is  9-!  millimeters,  the  magnifi- 
cation (which  is  the  ratio  between  size  of  object  and  image)  must  be 
9f  -T-  yq-  =  47.  That  is,  the  magnification  is  47  diameters,  or  47  times 
linear.     If  the  fractional  numbers  in  the  above  example  trouble  the  stu- 


Fmage 


Object- 
Fig.  93. 


Objective 


Objecl-b 
Object-a 


Fig.  9}. 


Figs.  93-94.  Figures  showing  that  the  size  of  object  and  image  vary  directly  as 
their  distance  from  the  center  of  the  lens.  In  Fig.  94  one  can  also  see  why  it  is 
necessary  to  focus  down,  i.  e.,  bring  ihe  object  and  objective  nearer  together  when 
the  tube  is  lengthened. 


CH.  TV.]  M.U.'.xrFf CATION  AND  MICROMETRY.  97 

dent,  both  may  be  reduced  to  the  same  denomination,  thus:  If  the  size 
of  the  image  is  found  to  be  9'-'  mm.  this  number  may  be  reduced  to 
tenths  mm.,  so  it  will  be  of  the  same  denomination  as  the  object.  In  9 
mm.  there  are  90  tenths,  and  in  f  there  are  4  tenths,  then  the  whole 
Length  of  the  image  is  90  -f-  4  =  94  tenths  of  a  millimeter.  The  object 
is  2  tenths  of  a  millimeter,  then  there  must  have  been  a  magnification 
of  94  -T-  2  =  47  diameters  in  order  to  produce  an  image  94  tenths  of  a 
millimeter  long. 

Put  the  25  mm.  (1  in.  C,  or  No.  4)  ocular  in  place  of  one  of  50  mm. 
focus,  and  then  put  the  camera  lucida  in  position.  Measure  the  size 
of  the  image  with  dividers  and  a  rule  as  before.  The  power  will  be 
considerably  greater  than  when  the  low  ocular  was  used.  This  is  be- 
cause the  virtual  image  (Fig.  21 ,  B3AS)  seen  with  the  high  ocular  is 
larger  than  the  one  seen  with  the  low  one.  The  real  image  "(Fig.  21, 
A'B1)  remains  nearly  the  same,  and  would  be  just  the  same  if  positive, 
par- focal  oculars  (§  34,  68,  note)  were  used. 

Lengthen  the  tube  of  the  microscope  50-60  mm.  by  pulling  out  the 
draw-tube.  Remove  the  camera  lucida,  and  focus,  then  replace  the 
camera  and  obtain  the  magnification.  It  will  be  greater  than  with  the 
shorter  tube.  This  is  because  the  real  image  (Fig.  94)  is  formed  far- 
ther from  the  objective  when  the  tube  is  lengthened,  and  the  objective 
must  be  brought  nearer  the  object  (Fig.  94).  The  law  is  :  The  size  of 
object  and  image  varies  directly  as  their  distance  from  the  center  of  the 
lens.     The  truth  of  this  statement  is  illustrated  by  Figs.  93  and  94. 

§  152.  Varying  the  Magnification  of  a  Compound  Microscope. 
It  will  be  seen  from  the  above  experiments  (§  151)  that  independently 
of  the  distance  at  which  the  microscopic  image  is  measured  (§  153 
there  are  three  ways  of  varying  the  power  of  a  compound  microscope. 
These  are  named  below  in  the  order  of  desirability. 
( 1 )  By  using  a  higher  or  lotver  objective. 

(2)  By  using  a  higher  or  lower  ocular. 

(3)  By  lengthe?iing  or  shortening  the  tube  of  the  microscope  (Fig.  94).* 
§  x53-   Standard  Distance  of  250  Millimeters  at  which  the  Vir- 
tual Image  is  Measured. — For  obtaining  the  magnification  of  both 

*  Amplifier. — In  addition  to  the  methods  of  varying  the  magnification  given  in 
\  152,  the  magnification  is  sometimes  increased  by  the  use  of  an  amplifier,  that  is 
a  diverging  lens  or  combination  placed  between  the  objective  and  ocular  and  serv- 
ing to  give  the  image-forming  rays  from  the  objective  an  increased  divergence. 
An  effective  form  of  this  accessory  was  made  by  Tolles,  who  made  it  as  a  small 
achromatic  concavo-convex  lens  to  be  screwed  into  the  lower  end  of  the  draw-tube 
(frontispiece)  and  thus  but  a  short  distance  above  the  objective.  The  divergence 
given  to  the  rays  increases  the  size  of  the  real  image  about  two-fold. 
7 


98 


MAGNIFICATION  AND  MICROMETRY. 


[CH.  IV. 


Fig.  95.  Figure  showing  the 
position  of  the  microscope,  the 
camera  lucida,  and  the  eye,  and 
the  different  sizes  of  the  image 
depending  upon  the  distance  at 
which  it  is  projected  from  the 
eye.  (a)  The  size  at  25  cm.; 
{b)  at 35  cm.,  (§  153). 


the  simple  and  the  compound  microscope  the  directions  were  to  measure 
the  virtual  image  at  a  distance  of  250  millimeters.  This  is  not  that  the 
image  could  not  be  seen  and  measured  at  any  other  distance,  but  be- 


vyl 

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Q     \         \         \ 

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u 

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1    \  1 
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J?~*&k**^~- ...  ..-.-.-  : ..  -,-  -K^tc^*--  ■'-:^. 

Fig.  96.  Sectional  view  of 
the  Abbe  Camera  Lucida  to 
show  that  in  measuring  the 
standard  distance  of  230 
millimeters,  one  must  meas- 
ure along  the  axis  from  the 
point  R,  at  the  left  of  the 
prism,  to  the  mirror,  and 
from  the  mirror  to  the 
drawing  surface.  For  a 
full  explanation  of  this 
camera  lucida,  see  next 
chapter,  (Figs.  102,  106). 


Fig.  96. 

cause  some  standard  must  be  selected,  and  this  is  the  most  common 
one.  The  necessity  for  the  adoption  of  some  common  standard  will  be 
seen  at  a  glance  in  Fig.  95,  where  is  represented  graphically  the  fact 
that  the  size  of  the  virtual  image  depends  directly  on  the  distance  at 
which  it  is  projected,  and  this  size  is  directly  proportional  to  the  verti- 
cal distance  from  the  apex  of  the  triangle,  of  which  it  forms  a  base. 


ci  i.  /r.] 


MAGNIFICATION  AND  MICROMETRY. 


99 


The  distance  of  250  millimeters  has  been  chosen  on  the  supposition  that 
it  is  the  distance  of  most  distinct  vision  for  the  normal  human  eye: 

Demonstrate  the  difference  in  magnification  due  to  the  distance  at 
which  the  image  is  projected,  by  raising  the  microscope  so  that  the  dis- 
tance will  be  350  millimeters,  then  lowering  to  150  millimeters. 

In  preparing  drawings  it  is  often  of  great  convenience  to  make  them 
at  a  distance  somewhat  less  or  somewhat  greater  than  the  standard.  In 
such  a  case  the  magnification  must  be  determined  for  the  special  dis- 
tance.     (See  the  next  chapter. ) 

For  discussions  of  the  magnification  of  the  microscope,  see  :  Beale, 
pp.  41,  355  ;  Carpenter-Dallinger,  pp.  26,  238;  Nageli  and  Schwen- 
dener,  p.  176,  ;  Ranvier,  p.  29;  Robin,  p.  126;  Amer.  Soc.  Micrs., 
1884,  p.  183  ;  1889,  p.  22  ;  Amer.  Jour.  Arts  and  Sciences,  1890,  p. 
50;  Jour.  Roy.  Micr.  Soc,  1888,  1889. 

§  154.  Table  of  Magnifications  and  of  the  Valuations  of  the 
Ocular  Micrometer. —  The  following  table  should  be  filled  out  by  each 
shident.  In  using  it  for  Micrometry  and  Draiving  it  is  necessary  to  keep 
clearly  in  mind  the  exact  conditions  under -which  the  determinations  were 
made,  and  also  the  ways  in  which  variatio?i  in  magnificatio7i  a?id  the  val- 
uation of  the  ocular  micrometer  may  be  produced  (§  152,  153,  163,  166). 


OCULAR                                  OCULAR 
50  nun.                                          25  mm. 

Objective. 

Tube 
in 

Tube 
out 

MM. 

Tube 

in 

Tube 
out 

MM. 

Ocular  Micrometer 

Valuation. 
tube  in.    out mm. 

X 

X 

X 

X 

X               X 

X               X 

X 

X 

X              X 

X 

X 

X 

X               X 

X 

X 

X              X 

X               X 

Simple  Microscope.            X 

IOO  MAGNIFICATION  AND  MICROMETRY.  [CH.  IV. 

MICROMETRY. 

§  155.  Micrometry  is  the  determination  of  the  size  of  objects  by  the 
aid  of  a  microscope. 

MICROMETRY   WITH   THE  SIMPLE   MICROSCOPE. 

§  156.  With  a  simple  microscope  (A),  the  easiest  and  best  way  is  to 
use  dividers  and  then  the  simple  microscope  to  see  when  the  points  of 
the  dividers  exactly  include  the  object.  The  spread  of  the  dividers  is 
then  obtained  as  above  (§  148).  This  amount  will  be  the  actual  size  of 
the  object,  as  the  microscope  was  only  used  in  helping  to  see  when  the 
divider  points  exactly  enclosed  the  object,  and  then  for  reading  the 
divisions  on  the  rule  in  getting  the  spread  of  the  dividers. 

(B)  One  may  put  the  object  under  the  simple  microscope  and  then, 
as  in  determining  the  power  (§  147),  measure  the  image  at  the  standard 
distance.  If  now  the  size  of  the  image  so  measured  is  divided  by  the 
magnification  of  the  simple  microscope,  the  quotient  will  give  the  actual 
size  of  the  object. 

Use  a  fly's  wing,  or  some  other  object  of  about  that  size,  and  try  to 
determine  the  width  in  the  two  ways  described  above.  If  all  the  work 
is  accurately  done  the  results  will  agree. 

MICROMETRY   WITH    THE    COMPOUND    MICROSCOPE. 

There  are  several  ways  of  varying  excellence  for  obtaining  the  size 
of  objects  with  the  compound  microscope,  the  method  with  the  ocular 
micrometer  (§  166-167)  being  most  accurate. 

§  157.  Unit  of  Measure  in  Micrometry. — As  most  of  the  objects 
measured  with  the  compound  microscope  are  smaller  than  any  of  the 
originally  named  divisions  of  the  meter,  and  the  common  or  decimal 
fractions  necessary  to  express  the  size  are  liable  to  be  unnecessarily 
cumbersome,  Hurting,  in  his  work  on  the  microscope  (1859),  proposed 
the  one  thousandth  of  a  millimeter  (y^n  mm.  or  0.001  mm.)  or 
one  millionth  of  a  meter  C 1  Tr"o"^"i7"0""(j  or  o. 00000 1  meter)  as  the  unit.  He 
named  this  unit  micro-millimeter  and  designated  it  mmm.  In  1869, 
Listing  (Carl's  Repetorium  fur  Experimental-Physik,  Bd,  X,  p.  5) 
favored  the  thousandth  of  a  millimeter  as  unit  and  introduced  the  name 
Mikron  or  micrum.  In  English  it  is  most  often  written  Micron,  plural 
micro,  or  microns,  pronunciation  Mic'rSn  or  Mic'rSn.  By  universal  con- 
sent the  sign  or  abbreviation  used  to  designate  it  is  the  Greek  /x.    Adopt- 


CII.  I V.  ]  MA  GNIFICA  TION  A  ND  MIC  ROME  TR  Y.  I  o  I 

ing  this  unit  and  sign,  one  would  express  five  thousandths  of  a  milli- 
meter (t^Vtt  or  o.oosths  mm.  )  thus,  5/x.::: 

\  158.  Micrometry  by  the  use  of  a  stage  micrometer  on  which  to  mount  the  ob- 
ject.—  In  this  method  the  object  is  mounted  on  a  micrometer  and  then  put  under 
the  microscope,  and  the  number  of  spaces  covered  by  the  object  is  read  off  directly. 
It  is  exactly  like  putting  any  large  object  on  a  rule  and  seeing  how  many  spaces  of 
the  rule  it  covers.  The  defect  in  the  method  is  that  it  is  impossible  to  properly 
arrange  objects  on  the  micrometer.  Unless  the  objects  are  circular  in  outline  they 
are  liable  to  be  oblique  in  position,  and  in  every  case  the  end  or  edges  of  the  object 
may  be  in  the  middle  of  a  space  instead  of  against  one  of  the  lines,  consequently 
the  size  must  lie  estimated  or  guessed  at  rather  than  really  measured. 

>;  [59.  Micrometry  by  dividing  the  size  of  the  image  by  the  magnifi- 
cation of  the  uiicroscope. — For  example,  employ  the  3  mm.  (y&  in.)  ob- 
jective, 25  mm.  ( 1  in. )  ocular,  and  a  Necturus'  red  blood-corpuscle  prepa- 
ration as  object.  Obtain  the  size  of  the  image  of  the  long  and  short  axes 
of  three  corpuscles  with  the  camera  lucida  and  dividers,  exactly  as  in  ob- 
taining the  magnification  of  the  microscope  (§  151 ).  Divide  the  size  of 
the  image  in  each  case  by  the  magnification,  and  the  result  will  be  the 
actual  size  of  the  blood-corpuscles.  Thus,  suppose  the  image  of  the 
long  axis  of  the  corpuscle  is  18  mm.  and  the  magnification  of  the  micro- 
scope 400  diameters  (§  145),  then  the  actual  length  of  this  long  axis  of 
the  corpuscle  is  18  mm.  ~r-  400  =  .045  mm.  or  45  /x  (§  157). 


FlG.  97.  Preparation  of  blood  with  a  ring 
around  a  group  of  blood  corpuscles. 


As  the  same  three  blood-corpuscles  are  to  be  measured  in  three  ways, 
it  is  an  advantage  to  put  a  delicate  ring  around  a  group  of  three  or  more 
corpuscles,  and  make  a  sketch  of  the  whole  enclosed  group,  marking  on, 
the  sketch  the  corpuscles  measured.  The  different  corpuscles  vary  con- 
siderably in  size,  so  that  accurate  comparison  of  different  methods  of. 
measurement  can  only  be  made  when  the  same  corpuscles  are  measured, 
in  each  of  the  ways  (Figs.  61-66). 


*The  term  Micromillimeter  ab.  mmm.  is  very  cumbersome,  and  besides  is  en- 
tirely inappropriate  since  the  adoption  of  definite  meanings  for  the  prefixes  mie.io 
and  mega,  meaning  respectively  one  millionth  and  one  million  times  the  unit  be- 
fore which  it  is  placed.  A  micromillimeter  would  then  mean  one-millionth  of  a. 
millimeter,  not  one  thousandth.  The  term  micron  has  been  adopted  by  the  great 
microscopical  societies,  the  international  commission  on  weights  and  measures, 
and  by  original  investigators,  and  is,  in  the  opinion  of  the  writer,  the  best  term  to 
employ.     Jour.  Roy.  Micr.  Soc,  1888,  p.  502  ;  Nature,  Vol.  XXXVII  (1888),  p.  388* 


1 02  MA  GNIFICA  TION  AND  MICRO  ME  TR  Y.  [  C H.  I V. 

§  160.  Micrometry  by  the  use  of  a  Stage  Micrometer  and  a  Camera 
Lucida. — Employ  the  same  object,  objective  and  ocular  as  before.  Put 
the  camera  lucida  in  position,  and  with  a  lead  pencil  make  dots  on  the 
paper  at  the  limits  of  the  image  of  the  blood-corpuscle.  Measure  the 
same  three  that  were  measured  in  §  159. 

Remove  the  object,  place  the  stage  micrometer  under  the  microscope, 
focus  well,  and  draw  the  lines  of  the  stage  micrometer  so  as  to  include 
the  dots  representing  the  limits  of  the  part  of  the  image  to  be  measured. 
As  the  value  of  the  spaces  on  the  stage  micrometer  is  known,  the  size 
of  the  object  is  determined  by  the  number  of  spaces  of  the  micrometer 
required  to  include  it. 

This  simply  enables  one  to  put  the  image  of  a  fine  rule  on  the  image 
of  a  microscopic  object.  It  is  theoretically  an  excellent  method,  and 
nearly  the  same  as  measuring  the  spread  of  the  dividers  with  a  simple 
microscope  (§  148,  167). 

OCULAR   MICROMETER. 

§  161.  Ocular  Micrometer,  Eye-Piece  Micrometer. — This,  asthe 
name  implies,  is  a  micrometer  to  be  used  with  the  ocular.  It  is  a  mi- 
crometer on  glass,  and  the  lines  are  sufficiently  coarse  to  be  clearly  seen 
by  the  ocular.  The  lines  should  be  equidistant  and  about  TVth  or  ^th 
mm.  apart,  and  ever}-  fifth  line  should  be  longer  and  heavier  to  facili- 
tate counting.  If  the  micrometer  is  ruled  in  squares  {net-micrometer} 
it  will  be  very  convenient  for  many  purposes. 

The  ocular  micrometer  is  placed  in  the  ocular,  no  matter  what  the 
form  of  the  ocular  (z.  e. ,  whether  positive  or  negative)  at  the  level  at 
which  the  real  image  is  formed  by  the  objective,  and  the  image  appears 
to  be  immediately  upon  or  under  the  ocular  micrometer,  and  hence  the 
number  of  spaces  on  the  ocular  micrometer  required  to  measure  the  real 
image  may  be  read  off  directly.  This  is  measuring  the  size  of  the  real 
image,  however,  and  the  actual  size  of  the  object  can  only  be  deter- 
mined by  determining  the  ratio  between  the  size  of  the  real  image  and 
the  object.  In  other  words,  it  is  necessary  to  get  the  valuation  of  the 
ocular  micrometer  in  terms  of  a  stage  micrometer. 

§  162.  Valuation  of  the  Ocular  Micrometer. — This  is  the  value 
of  the  divisions  of  the  ocular  micrometer  for  the  purposes  of  microm- 
etry, and  is  entirely  relative,  depending  upon  the  magnification  of  the 
real  image  formed  by  the  objective,  consequently  it  changes  with  ever}' 
change  in  the  magnification  of  the  real  image,  and  must  be  specially 
determined  for  every  optical  combination  (z.  e.,  objective  and  ocular), 


CH.  IV.} 


M  ACS //■'/(:  I  VVOX  AND  MICROMETRY. 


103 


and  for  every  change  in  the  length  of  tin- tube  of  the  microscope.  That 
is,  it  is  necessary  to  determine  the  ocular  micrometer  valuation  for  every 
condition  modifying  the  real  image  of  the  microscope  (§  152 

Any  I  Invgenian  ocular  (Fig  30)  may,  however,  be  used  as  a  micrometer  ocular 
by  placing  the  ocular  micrometer  at  the  level  of  the  ocular  diaphragm,  where  tin- 
real  image  is  formed.  It'  there  is  a  slit  in  the  side-  of  the  ocular,  and  the  ocular 
micrometer  is  mounted  in  some  way  it  may  be  introduced  through  the  opening  in 
the  side.  When  no  side  opening  exists  the  mounting  of  the  eye-lens  may  be  un- 
screwed and  the  ocular  micrometer,  if  on  a  cover-glass,  can  be  laid  on  the  upp<  t 
side  of  the  ocular  diaphragm. 


Figs.  98,  99.  Filar  Ocular  Micrometer  with  Field  1  Bausch  &  Lomb,  Optical 
Co. ).     For  other  ocular  micrometers  see  />/>.  23,  26. 

'</.  163.  Obtaining  the  Valuation  of  the  Filar  Micrometer.  This  micrometer 
1  Fig.  98-99)  consists  of  a  Ramsden's  ocular  and  cross  lines.  As  seen  in  Fig.  98 
there  are  three  lines.  The  horizontal  and  one  vertical  line  are  fixed.  One  vertical 
line  may  he  moved  by  the  screw  hack  and  forth  across  the  field. 

For  obtaining  the  valuation  of  this  ocular  micrometer  an  accurate  stage  mi- 
crometer must  be  used.  Carefully  focus  the  ,,',,111  mm.  spaces.  The  lines  of  the 
ocular  micrometer  should  also  be  sharp.  It'  they  are  not  focus  them  by  moving 
the  top  of  the  ocular  up  or  down  ( \  164  |.  Make  the  vertical  lines  of  the  filar  mi- 
crometer parallel  with  the  Hues  of  the  stage  micrometer.  Take  the  precautions 
regarding  the  width  of  the  stage  micrometer  lines  given  in  \  167  (see  also  Fig. 
101).  Note  the  position  of  the  graduated  wheel  and  of  the  teeth  of  th<  recording 
comb,  and  then  rotate  the  wheel  until  the  movable  line  traverse-  one  space  on  the 
Stage  micrometer.  Each  tooth  of  the  recording  comb  indicates  a  total  revolution 
of  the  wheel,  and  by  noting  the  number  of  teeth  required  and  the  graduations  on 
the  wheel,  the  revolutions  and  parts  of  revolutions  required  to  measure  the 
mm.  of  the  stage  micrometer  can  be  easily  noted.       Measure  in  like  manner   1  Or  5 

spaces  and  get  the  average.     Suppose  this  average  is  i'4th   revolutions  or  1  25 
graduations  on  the  wheel,  to  measure  the  |,',„th  mm.  or   iom  I  see  '.hen  one 

of    the  graduations   on    the   wheel    would    measure    [O/i  divided    bj    1  -\S        .08/t.      In 


104  MAGNIFICATION  AND  MICROMETRY.  [CH.  IV. 

using  this  valuation  for  actual  measurement,  the  tube  of  the  microscope  and  the 
objective  must  be  exactly  as  when  obtaining  the  valuation  (see  #  165). 

Example  of  Measurement. — Suppose  one  uses  the  red  blood  corpuscles  of  a  dog 
or  monkey,  etc.,  every  condition  being  as  when  the  valuation  was  determined,  one 
notes  very  accurately  how  many  of  the  graduations  on  the  wheel  are  required  to 
make  the  movable  line  traverse  the  object  from  edge  to  edge.  Suppose  it  requires 
94  of  the  graduations  to  measure  the  diameter,  the  actual  size  of  the  corpuscle 
would  be  94  X  .08/*^=  7.52/x. 

The  advantage  of  the  filar  micrometer  is  that  the  valuation  of  one  graduation 
being  so  small,  even  the  smallest  object  to  be  measured  would  require  several 
graduations  to  measure  it.  In  ocular  micrometers  with  fixed  lines,  small  ob- 
jects like  bacteria  might  not  fill  even  one  space,  therefore  estimations,  not  meas- 
urements, must  be  made.  For  large  objects,  like  most  of  the  tissue  elements,  the 
ocular  micrometers  with  fixed  lines  answer  very  well,  for  the  part  which  must  be 
estimated  is  relatively  small,  and  the  chance  of  error  is  correspondingly  small. 

£  164.  Obtaining  the  Ocular  Micrometer  Valuation  for  an  Oc- 
ular Micrometer  with  Fixed  Lines  (Figs.  33,  34,  p.  25). — Use  the 
stage  micrometer  as  object.  Light  the  field  well  and  look  into  the  mi- 
croscope. The  lines  of  the  ocular  micrometer  should  be  very  sharply 
defined.  If  they  are  not  raise  or  lower  the  eye-lens  to  make  them  so  ; 
that  is,  focus  as  with  the  simple  magnifier. 

When  the  lines  of  the  ocular  micrometer  are  distinct,  focus  the  mi- 
croscope (§  45,  46,  56)  for  the  stage  micrometer.  The  image  of  the 
stage  micrometer  will  appear  to  be  directly  under  or  upon  the  ocular 
micrometer. 

Make  the  lines  of  the  two  mioometcrs  partxllel  by  rotating  the  ocular 
or  changing  the  position  of  the  stage  micrometer,  or  both  if  necessary, 
and  then  make  any  two  lines  of  the  stage  micrometer  coincide  with  any 
two  on  the  ocular  micrometer.  To  do  this  it  may  be  necessary  to  pull 
out  the  draw-tube  a  greater  or  less  distance.  See  how  many  spaces 
are  included  on  each  of  the  micrometers. 

Divide  the  value  of  the  included  space  or  spaces  on  the  stage  microm- 
eter by  the  number  of  divisions  on  the  ocular  micrometer  required  to 
include  them,  and  the  quotient  so  obtained  will  give  the  valuation  of 
the  ocular  micrometer  in  fractions  of  the  unit  of  measure  of  the  stage 
micrometer.  For  example,  suppose  the  millimeter  is  taken  as  the  unit 
for  the  stage  micrometer  and  this  unit  is  divided  into  spaces  of  yVtn  anc^ 
Y^th  millimeter.  If  now,  with  a  given  optical  combination  and  tube- 
length,  it  requires  10  spaces  on  the  ocular  micrometer  to  include  the 
real  image  of  TVth  millimeter  on  the  stage  micrometer,  obviously  one 
space  on  the  ocular  micrometer  would  include  only  one-tenth  as  much, 
or  yoth  mm.  -=-  10  =  yiyoth  mm.  That  is,  each  space  on  the  ocular  mi- 
crometer would  include  T| rffth  of  a  millimeter  on  the  stage  micrometer, 
oryj^th  millimeter  of  length  of  any  object  under  the  microscope,  the 
conditions  remaining  the  same.       Or,  in  other  words,  it  would  require 


CH.  IV.]  MA  i  tNIFICA  TION  AND  MICROME  TR  V.  1 05 

100  spaces  on  the  ocular  micrometer  to  include  1  millimeter  on  the  staj 
micrometer,  then  as  before  1  space  of  the  ocular  micrometer  would  have 
a  valuation  of  ,  /, , , 1 1 1  millimeter  for  the  purposesof  micrometry  ;  and  the 

size  of  any  minute  object  may  be  determined  by  multiplying  this  valua- 
tion of  one  space  by  the  number  of  spaces  required  to  include  it.  For 
example,  suppose  the  fly's  wing  or  some  part  of  it  covered  8  spaces  on 
the  ocular  micrometer,  it  would  be  known  that  the  real  size  of  the  part 
measured  is  [-forth  mm.    X   8  =   ,  *  „-  mm.  or  80  //.  (§  157). 

§  [65.  Varying  the  Ocular  Micrometer  Valuation. — Any  change 
in  the  objective,  the  ocular  or  the  tube-length  of  the  microscope,  that 
is  to  say,  any  change  in  the  size  of  the  real  image,  produces  a  corre- 
sponding change  in  the  ocular  micrometer  valuation  (§  152,  161). 

S  166.  Micrometry  with  the  Ocular  Micrometer. — Use  the  3  mm. 
( }i  in. )  objective  and  preparation  of  Necturus  blood-corpuscles  as  object. 
Make  certain  that  the  tube  of  the  microscope  is  of  the  same  length  as 
when  determining  the  ocular  micrometer  valuation.  In  a  word,  be  sure 
that  all  the  conditions  are  exactly  as  when  the  valuation  was  determined, 
then  put  the  preparation  under  the  microscope  and  find  the  same  three 
red  corpuscles  that  were  measured  in  the  other  ways  |  .*,  159,  160). 

Count  the  divisions  on  the  ocular  micrometer  required  to  enclose  or 
measure  the  long  and  the  short  axis  of  each  of  the  three  corpuscles, 
then  multiply  the  number  of  spaces  in  each  case  by  the  valuation  of  the 
ocular  micrometer  for  this  objective,  tube-length  and  ocular,  and  the 
results  will  represent  the  actual  length  of  the  axes  of  the  corpuscles  in 
each  case. 

The  same  corpuscle  is,  of  course,  of  the  same  actual  size,  when  meas- 
ured in  each  of  the  three  ways,  so  that  if  the  methods  are  correct  and 
the  work  carefully  enough  done,  the  same  results  should  be  obtained  by 
each  method.     See  general  remarks  on  micrometry  (§  167 ).* 

*  There  are  three  ways  of  using  the  ocular  micrometer,  or  of  arriving  at  the  size 
of  the  ohjects  measured  with  it  : 

(A)  By  finding  the  value  of  a  division  of  the  ocular  micrometer  for  each  optical 
combination  and  tube-length  used,  and  employing  this  valuation  as  a  multiplier. 
This  is  the  method  given  in  the  text,  and  is  the  one  most  frequently  employed. 
Thus,  suppose  with  a  given  optical  combination  and  tube-length  it  required  five 
divisions  on  the  ocular  micrometer  to  include  the  image  of  i-„ths  millimeter  of  the 
stage  micrometer,  then  obviously  one  space  on  the  ocular  micrometer  would  in- 
clude 1th  of  r.iths  mm.  or  J-th  mm.  ;  and  the  size  of  any  unknown  object  under 
the  microscope  would  be  obtained  by  multiplying  the  number  of  divisions  >>n  the 
ocular  micrometer  required  to  include  its  image  by  the  value  of  one  space,  or  in 
this  case,  2'Tth  mm.  Suppose  some  object,  as  the  fly's  wing,  required  15  spaces  of 
the  ocular  micrometer  to  include  some  part  of  it,  then  the  actual  size  of  this  part 
of  the  wing  would  be  15  X  /j  —  Iths,  or  0.6  mm. 


106  MAGNIFICATION  AND  MICROMETRY.  \CH.  IV. 

\  167.  Remarks  on  Micrometry. — In  using  adjustable  objectives  (§  22,  96),  the 
magnification  of  the  objective  varies  with  the  position  of  the  adjusting  collar,  be- 
ing greater  when  the  adjustment  is  closed  as  for  thick  cover  glasses  than  when 
open,  as  for  thin  ones.  This  variation  in  the  magnification  of  the  objective  pro- 
duces a  corresponding  change  in  the  magnification  of  the  entire  microscope  and 
the  ocular  micrometer  valuation— therefore  it  is  necessary  to  determine  the  mag- 
nification and  ocular  micrometer  valuation  for  each  position  of  the  adjusting  collar. 

While  the  principles  of  micrometry  are  simple,  it  is  very  difficult  to  get  the  ex- 
act size  of  microscopic  objects.     This  is  due  to  the  lack  of  perfection  and  uni- 

(B)  By  finding  the  number  of  divisions  on  the  ocular  micrometer  required  to  in- 
clude the  image  of  an  entire  millimeter  of  the  stage  micrometer,  and  using  this 
number  as  a  divisor.  This  number  is  also  sometimes  called  the  ocular  micrometer 
ratio.  Taking  the  same  case  as  in  (A),  suppose  five  divisions  of  the  ocular  mi- 
crometer are  required  to  include  the  image  of  T2ffths  mm.,  on  the  stage  micrometer, 
then  evidently  it  would  require  5  -5-  T2ff  =  25  divisions  on  the  ocular  micrometer  to 
include  a  whole  millimeter  on  the  stage  micrometer,  then  the  number  of  divisions 
of  the  ocular  micrometer  required  to  measure  an  object  divided  by  25  would  give 
the  actual  size  of  the  object  in  millimeters  or  in  a  fraction  of  a  millimeter.  Thus, 
suppose  it  required  15  divisions  of  the  ocular  micrometer  to  include  the  image  of 
some  part  of  the  fly's  wing,  the  actual  size  of  the  part  included  would  be  15-7-25 
=  f  or  0.6  mm.  This  method  is  really  exactly  like  the  one  in  (A),  for  dividing  by 
25  is  the  same  as  multiplying  by  ^th. 

(C)  By  having  the  ocular  micrometer  ruled  in  millimeters  and  divisions  of  a  mil- 
limeter, and  then  getting  the  size  of  the  real  image  in  millimeters.  In  employing 
this  method  a  stage  micrometer  is  used  as  object  and  the  size  of  the  image  of  one 
or  more  divisious  is  measured  by  the  ocular  micrometer,  thus  :  Suppose  the  stage 
micrometer  is  ruled  in  TVth  and  i^th  mm.  and  the  ocular  micrometer  is  ruled  in 
millimeters  and  y^tti  mm.  Taking  T3(Jth  mm.  on  the  stage  micrometer  as  object, 
as  in  the  other  cases,  suppose  it  requires  10  of  the  yts^  mm.  spaces  or  1  mm.  to 
measure  the  real  image,  then  the  real  image  must  be  magnified  j{J  -j-t2o  =  5  diame- 
ters, that  is,  the  real  image  is  five  times  as  great  in  length  as  the  object,  and  the 
size  of  an  object  may  be  determined  by  putting  it  under  the  microscope  and  getting 
the  size  of  the  real  image  in  millimeters  with  the  ocular  micrometer  and  dividing 
it  by  the  magnification  of  the  real  image,  which  in  this  case  is  5  diameters. 

Use  the  fly's  wing  as  object,  as  in  the  other  cases,  and  measure  the  image  of  the 
same  part.  Suppose  that  it  required  30  of  the  T\  mm.  divisions  =  f'§  mm  or  3  mm. 
to  include  the  image  of  the  part  measured,  then  evidently  the  actual  size  of  the 
part  measured  would  be  3  mm.  -4-  5  =  $  mm. ,  the  same  result  as  in  the  other  cases. 

In  comparing  these  methods  it  will  be  seen  that  in  the  first  two  (A  and  B)  the 
ocular  micrometer  may  be  simply  ruled  with  equidistant  lines  without  regard  to 
the  absolute  size  in  millimeters  or  inches  of  the  spaces.  In  the  last  method  the 
ocular  micrometer  must  have  its  spaces  some  known  division  of  a  millimeter  or 
inch.  In  the  first  two  methods  only  one  standard  of  measure  is  required,  viz  ,  the 
stage  micrometer  ;  in  the  last  method  two  standards  must  be  used, — a  stage  mi- 
crometer and  an  ocular  micrometer.  Of  course,  the  ocular  micrometer  in  the  first 
two  cases  must  have  the  lines  equidistant  as  well  as  in  the  last  case,  but  ruling  lines 
equidistant  and  an  exact  division  of  a  millimeter  or  an  inch  are  two  quite  different 
matters. 


CH.  IV.] 


MAGNIFICATION  AND  MICROMETRY. 


107 


fortuity  of  micrometers,  and  the  difficulty  of  determining  the  exact  limits  of  the 
object  to  be  measured.  Hence,  all  microscopic  measurements  are  only  approxi- 
mately correct,  the  error  lessening  with  the  increasing  perfection  of  the  apparatus 
and  the  skill  of  the  observer. 

A  difficulty  when  one  is  using  high  powers  is  the  width  of  the  lines  of  the  mi- 
crometer. If  the  micrometer  is  perfectly  accurate  half  the  width  of  each  line  be- 
longs to  the  contiguous  spaces,  hence  one  should  measure  the  image  of  the  space 
from  the  centers  of  the  lines  bordering  the  space,  or  as  this  is  somewhat  difficult 
in  using  the  ocular  micrometer,  one  may  measure  from  the  inside  of  one  border- 
ing line  and  from  the  outside  of  the  other.  If  the  lines  are  of  equal  width  this  is 
as  accurate  as  measuring  from  the  center  of  the  lines.  Evidently  it  would  not  be 
right  to  measure  from  either  the  inside  or  the  outside  of  both  lines  (  Fig.  101 ). 

It  is  also  necessary  in  micrometry  to  use  an  objective  of  sufficient  power  to  en- 
able one  to  see  all  the  details  of  an  object  with  great  distinctness.  The  necessity 
of  using  sufficient  amplification  in  micrometry  has  been  especially  remarked  upon 
by  Richardson,  Monthly  Micr.  Jour.,  1874,  1875  ;  Rogers,  Proc.  Amer.  Soc.  Micro- 
scopists,  1882,  p.  239;  Ewell,  North  American  Pract.,  1890,  pp.  97,  173. 


Fig.  ior.  77/i?  appearance  of  the  coarse 
stage  and  of  the  fine  ocular  micrometer 
lines  when  using  a  high  objective. 

(A).  The  method  of  measuring  the  spaces 
by  putting  the  fine  ocular  micrometer  lines 
opposite  the  center  of  the  coarse  stage  mi- 
crometer lines. 

(B).  Method  of  measuring  the  spaces  of 
the  stage  micrometer  by  putting  one  line 
of  the  ocular  micrometer  (o.m.)  at  the  in- 
side and  one  at  the  outside  of  the  coarse 
stage  micrometer  lines  (s.m. ). 


Fig.  ioi. 


As  to  the  limit  of  accuracy  in  micrometry,  one  who  has  justly  earned  the  right 
to  speak  with  authority,  expresses  himself  as  follows  :  "  I  assume  that  0.21.1  is  the 
limit  of  precision  in  microscopic  measures,  beyond  which  it  is  impossible  to  go 
with  certainty."     W.  A.  Rogers,  Proc.  Amer.  Soc   Micrs.,  1SS3,  p.  19S. 

In  comparing  the  methods  of  micrometry  with  the  compound  microscope,  given 
above  (15S,  159,  160,  166),  the  one  given  in  \  158  is  impracticable,  that  given  in 
\  159  is  open  to  the  objection  that  two  standards  are  required, — the  stage  microme- 
ter, and  the  steel  rule  ;  it  is  open  to  the  further  objection  that  several  different  ope- 
rations are  necessary,  each  operation  adding  to  the  probability  of  error.  Theoret- 
ically the  method  given  in  \  160  is  good,  but  it  is  open  to  the  very  serious  objection 
in  practice  that  it  requires  so  many  operations  which  are  especially  liable  to  intro- 
duce errors.  The  method  that  experience  has  found  most  safe  and  expeditious, 
and  applicable  to  all  objects,  is  the  method  with  the  ocular  micrometer.  If  the 
valuation  of  the  ocular  micrometer  has  been  accurately  determined,  then  the  only 
difficulty  is  in  deciding  on  the  exact  limits  of  the  object  to  be  measured  and  so  ar- 
ranging the  ocular  micrometer  that  these  limits  are  inclosed  by  some  divisions  of 


1 08  MA  GNIFICA  TION  AND  MICRO  ME  TR  Y.  [  CH.  IV. 

the  micrometer.  Where  the  object  is  not  exactly  included  by  whole  spaces  on  the 
ocular  micrometer,  the  chance  of  error  comes  in,  in  estimating  just  how  far  into  a 
space  the  object  reaches  on  the  side  not  in  contact  with  one  of  the  micrometer 
lines.  If  the  ocular  micrometer  has  some  quite  narrow  spaces,  and  others  consid- 
erably larger,  one  can  nearly  always  manage  to  exactly  include  the  object  by  some 
two  lines.  The  ocular  screw- micrometer  (Fig.  100)  obviates  this  entirely  as  the 
cross  hairs  or  lines  traverse  the  object  or  its  real  image,  and  whether  this  distance 
be  great  or  small  it  can  be  read  off  on  the  graduated  wheel,  and  no  estimation  or 
guess  work  is  necessary. 

For  those  especially  interested  in  micrometry,  as  in  its  relation  to  medical  juris- 
prudence, the  following  references  are  recommended.  These  articles  consider  the 
problem  in  a  scientific  as  well  as  a  practical  spirit  :  The  papers  of  Prof.  Wm.  A. 
Rogers  on  micrometers  and  micrometry,  in  the  Amer.  Ouar.  Micr.  Jour. ,  Vol.  I, 
pp.  97,  208;  Proceedings  Amer.  Soc.  Microscopists,  1882,  1883,  18S7.  Dr.  M.  D. 
Fwell,  Proc.  Amer.  Soc.  Micrs.,  1890;  The  Microscope,  18S9,  pp.  43-45;  North 
Amer.  Pract,  1890,  pp.  97,  173.  Dr.  J.  J.  Woodward,  Amer.  Jour,  of  the  Med.  Sci., 
1875.  M.  C.  White,  Article  Blood-stains,  Ref.  Hand-Book,  Med.  Sciences,  1885. 
Medico-Legal  Journal,  Vol.  XII.  For  the  change  in  magnification  due  to  a  change 
in  the  adjustment  of  adjustable  objectives,  see  Jour.  Roy.  Micr.  Soc,  1880,  p.  702  ; 
Amer.  Monthly  Micr.  Jour.,  1880,  p.  67. 

If  one  consults  the  medico  legal  journals,  the  Index  Medicus,  and  the  Index 
Catalog  of  the  Library  of  the  Surgeon  General's  Office,  under  Micrometry,  Blood, 
and  Jurisprudence,  he  can  get  on  track  of  the  main  work  which  has  been  and  is  be- 
ing done. 


CHAPTER  Y 


DRAWING  WITH  THE  MICROSCOPE. 


APPARATUS   AND   MATERIAL   FOR   THIS   CHAPTER. 

Microscope,  Abbe  camera  lucida,  drawing  board  thumb  tacks,  pencils,  paper, 
and  microscope  screen  (Fig.  58). 

DRAWING    MICROSCOPIC    OBJECTS. 

§  168.  Microscopic  objects  may  be  drawn  free-hand  directly  from  the 
microscope,  but  in  this  way  a  picture  giving  only  the  general  appear- 
ance and  relations  of  parts  is  obtained.  For  pictures  which  shall  have 
all  the  parts  of  the  object  in  true  proportions  and  relations,  it  is  neces- 
sary to  obtain  an  exact  outline  of  the  image  of  the  object,  and  to  locate 
in  this  outline  all  the  principal  details  of  structure.  It  is  then  possible 
to  complete  the  picture  free-hand  from  the  appearance  of  the  object  un- 
der the  microscope.  The  appliance  used  in  obtaining  outlines,  etc.,  of 
the  microscopic  image  is  known  as  a  camera  lucida. 

§  169.  Camera  Lucida. — This  is  an  optical  apparatus  for  enabling 
one  to  see  objects  in  greatly  different  situations,  as  if  in  one  field  of  vis- 
ion, and  with  the  same  eye.  In  other  words,  it  is  an  optical  device  for 
superimposing  or  combining  two  fields  of  view  in  one  eye. 

As  applied  to  the  microscope,  it  causes  the  magnified  virtual  image 
of  the  object  under  the  microscope  to  appear  as  if  projected  upon  the 
table  or  drawing  board,  where  it  is  visible  with  the  drawing  paper,  pen- 
cils, dividers,  etc.,  by  the  same  eye,  and  in  the  same  field  of  vision. 
The  microscopic  image  appears  like  a  picture  on  the  drawing  paper. 
This  is  accomplished  in  two  distinct  ways  : 

(A)  By  a  camera  lucida  reflecting  the  rays  from  the  microscope  so 
that  their  direction  when  they  reach  the  eye  coincides  with  that  of  the 
rays  from  the  drawing  paper,  pencils,  etc.  In  some  of  the  camera  luci- 
das  of  this  group  (Wollaston's,  Fig.  105),  the  rays  are  reflected  twice, 
and  the  image  appears  as  when  looking  directly  into  the  microscope. 
In  others  the  rays  are  reflected  but  once,  and  the  image  has  the  inver- 
sion produced  by  a  plane  mirror.  For  drawing  purposes  this  inversion 
is  a  great  objection,  as  it  is  necessary  to  similarly  invert  all  the  details 
added  free-hand. 


I  IO 


DRA  WING  WITH  THE  MICROSCOPE. 


[CH    V. 


(B)  By  a  camera  lucida  reflecting  the  rays  of  light  from  the  drawing 
paper,  etc.,  so  that  their  direction  when  they  reach  the  eye  coincides 
with  the  direction  of  the  rays  from  the  microscope  (Fig.  57,  60).  In 
all  of  the  camera  lucidas  of  this  group,  the  rays  from  the  paper  are  twice 
reflected  and  no  inversion  appears. 

The  better  forms  of  camera  lucidas  (Wollaston's,  Grunow's,  Abbe's, 
etc.),  may  be  used  for  drawing  both  with  low  and  with  high  powers. 
Some  require  the  microscope  to  be  inclined  (Fig.  105),  while  others  are 

V 


Fig.  101 


Fig.  104. 


Fig.  102. 


Fig.  102.  Abbe  Camera  Lucida  with  the 
mirror  at  45°,  the  drawing  surface  hori- 
zontal, and  the  microscope  vertical. 

Axis,  Axis.  Axial  ray  from  the  mi- 
croscope and  from  the  drawing  surface. 
A  B.  Marginal  rays  of  the  field  on  the 
drawing  surface,  a  b.  Sectional  view  of 
the  silvered  surf  ace  on  the  upper  of  the  tri- 
angular prisms  composing  the  cubical 
prism  ( P).  The  silvered  surface  is  shown 
as  incomplete  in  the  center,  thus  giving  passage  to  the  rays  from  the  microscope. 
Foot.    Foot  or  base  of  the  microscope. 

G.  Smoked  glass  seen  in  section.     It  is  placed  between  the  mirror  and  the  prism 
to  reduce  the  light  from  the  drawing  surface. 

Mirror.   The  mirror  of  the  camera  lucida.     A  quadrant  (Q)  has  been  added  to 
indicate  the  angle  of  inclination  of  the  mirror,  which  in  this  case  is  45°. 

Ocular.    Ocular  of  the  microscope  over  which  the  pristn  of  the  camera  lucida  is 
placed. 

P,  P.  Drawing  pencil  and  the  cubical  prism  over  the  ocular. 

Fig.  103.  Geometrical  figure  showing  the  angles  made  by  the  axial  ray  with  the 
drawing  surface  and  the  mirror. 
A  B.   The  drawing  surface. 

Fig.  104.  Ocular  showing  eye-point,  E  P.     It  is  at  this  point  both  horizontally 
andvertically  that  the  hole  in  the  silvered  surface  should  be  placed  {I  173). 


CI  I.    /'] 


DRAWING  Willi  THE  MICROSCOPE. 


i  i  i 


designed  to  be  used  on  the  microscope  in  a  vertical  position.  As  in  bio- 
logical work,  it  is  often  necessary  to  have  the  microscope  vertical,  the 
form  for  a  vertical  microscope  is  to  be  preferred  ;  but  see  Figs.  102-i  1 1 . 
§  170.  Avoidance  of  Distortion. — /;/  order  that  the  picture  drawn 
by  the  aid  of  a  camera  lucida  may  not  be  distorted^  it  is  necessary  that  the 
axial  ray  from  the  image  on  the  drawing  surface  shall  be  at  right  angles 

to  the  drawing  surface  (Figs.  102,  105,  106). 

\  171.  Wollaston's  Camera  Lucida. — This  is  a  quadrangular  prism  of  glass  put 
in  the  path  of  the  rays  from  the  microscope,  and  it  serves  to  change  the  direction 
of  the  axial  ray  93  degrees.  In  using  it  the  microscope  is  made  horizontal,  and  the 
rays  from  the  microscope  enter  one  half  of  the  pupil  while  rays  from  the  drawing 
surface  enter  the  other  half  of  the  pupil.  As  seen  in  the  figure  (Fig.  105),  the  fields 
partly  overlap,  and  where  they  do  so  overlap,  pencil  or  dividers  and  microscopic 
image  can  be  seen  together. 

In  drawing  or  using  the  dividers  with  the  Wollaston  camera  lucida  it  is  necessary 
to  have  the  field  of  the  microscope  and  the  drawing  surface  about  equally  lighted. 
If  the  drawing  surface  is  too  brilliantly  lighted  the  pencil  or  dividers  may  be  seen 
very  clearly,  but  the  microscopic  image  will  be  obscure.  On  the  other  hand,  if  the 
field  of  the  microscope  has  too  much  light  the  microscopic  image  will  be  very  defi- 
nite, but  the  pencil  or  dividers  will  not  be  visible.  It  is  necessary,  as  with  the 
Abbe  camera  lucida  (|  173),  to  have  the  Wollaston  prism  properly  arranged  with 
reference  to  the  axis  of  the  microscope  and  the  eye-point.  If  it  is  not,  one  will  be 
unable  to  see  the  image  well,  and  may  be  entirely  unable  to  see  the  pencil  and  the 
image  at  the  same  time.  Again,  as  rays  from  the  microscope  and  from  the  draw- 
ing surface  must  enter  independent  parts  of  the  pupil  of  the  same  eye,  one  must 
hold  the  eye  so  that  the  pupil  is  partly  over  the  camera  lucida  and  partly  over  the 
drawing  surface.  One  can  1  ell  the  proper  position  by  trial.  This  is  not  a  very  sat- 
isfactory camera  to  draw  with,  but  it  is  a  very  good  form  to  measure  the  vertical 
distance  of  250  mm.  at  which  the  drawing  surface  should  be  placed  when  determin- 
ing magnification  ($  153). 

Fig.  105.  Wollaston' s  Camera  Lu- 
cida, showing  the  rays  from  the  mi- 
croscope and  from  the  drawing  sur- 
face, and  the  position  of  the  pupil  of 
the  eye. 

For  full  explanation  see  Fig.  g2. 

$  172.  Abbe  Camera  Luci- 
da.— This  consists  of  a  cube  of 
glass  cut  into  two  triangular 
prisms  and  silvered  on  the  upper 
one.  A  small  oval  hole  is  then 
cut  out  of  the  center  of  the  sil- 
vered surface  and  the  two  prisms 
are  cemented  together,  thus  giv- 
ing a  cubical  prism  with  a  per- 
forated 45  degree  mirror  (Fig. 


112  DRAWING  WITH  THE  MICROSCOPE.  [CH.   V. 

102,  ab).  The  upper  surface  of  the  prism  is  covered  by  a  perforated 
metal  plate  (Fig.  108).  This  prism  is  placed  over  the  ocular  in  such  a 
way  that  the  light  from  the  microscope  passes  through  the  hole  in  the 
silvered  face  and  thence  directly  to  the  eye.  Light  from  the  drawing 
surface  is  reflected  by  a  mirror  to  the  silvered  surface  of  the  prism  and 
reflected  by  this  surface  to  the  eye  in  company  with  the  rays  from  the 
microscope,  so  that  the  two  fields  appear  as  one,  and  the  image  is  seen 
as  if  on  the  drawing  surface  (Figs.  102,  106).  It  is  designed  for  use 
with  a  vertical  microscope,  but  see  §  174. 

§  173.  Arrangement  of  the  Camera  Lucida  Prism. — In  placing 
this  camera  lucida  over  the  ocular  for  drawing  or  the  determination  of 
magnification,  the  center  of  the  hole  in  the  silvered  surface  is  placed  in 
the  optic  axis  of  the  microscope.  This  is  done  by  properly  arranging 
the  centering  screws  that  clamp  the  camera  to  the  microscope  tube  or 
ocular.  The  perforation  in  the  silvered  surface  must  also  be  at  the  level 
of  the  eye-point  (Fig.  104).  In  other  words,  the  prism  must  be  so 
arranged  vertically  and  horizontally  that  the  hole  in  the  silvered  surface 
will  be  in  the  axis  of  the  microscope  and  co-incident  with  the  eye-point 
of  the  ocular.  If  it  is  above  Or  below,  or  to  one  side  of  the  eye-point, 
part  or  all  of  the  field  of  the  microscope  will  be  cut  off.  As  stated 
above,  the  centering  screws  are  for  the  proper  horizontal  arrangement 
of  the  prism.  The  prism  is  set  at  the  right  height  by  the  makers  for 
the  eye-point  of  a  medium  ocular.  If  one  desires  to  use  an  ocular  with 
the  eye-point  farther  away  or  nearer,  as  in  using  high  or  low  oculars, 
the  position  of  the  eye-point  may  be  determined  as  directed  in  §  55  and 
the  prism  loosened  and  raised  or  lowered  to  the  proper  level  ;  but  in 
doing  this  one  should  avoid  setting  the  prism  obliquely  to  the  mirror. 

In  the  latest  and  best  forms  of  this  camera  lucida  special  arrangements 
have  been  made  for  raising  or  lowering  the  prism  so  that  it  may  be  used 
with  equal  satisfaction  on  oculars  with  the  eye-point  at  different  levels, 
and  the  prism  is  hinged  to  turn  aside  without  disturbing  the  mirror. 

See  the  latest  catalogs  of  Zeiss,  L,eitz,  and  the  Bausch  &  L,omb  Op- 
tical Co. 

One  can  determine  when  the  camera  is  in  a  proper  position  by  looking 
into  the  microscope  through  it.  If  the  field  of  the  microscope  appears 
as  a  circle  and  of  about  the  same  size  as  without  the  camera  lucida,  then 
the  prism  is  in  a  proper  position.  If  one  side  of  the  field  is  dark,  then 
the  prism  is  to  one  side  of  the  center  ;  if  the  field  is  considerably  smaller 
than  when  the  prism  is  turned  off  the  ocular,  it  indicates  that  it  is  not 
at  the  correct  level,  i.  c,  it  is  above  or  below  the  eye-point. 

§  174.    Arrangement  of  the  Mirror  and  the  Drawing  Surface. — 


CH.   /',]  DRAWING  WITH  THE  MICROSCOPE.  113 

The  Abbe  camera  lucida  was  designed  for  use  with  a  vertical  microso  ■ 
(Fig.  102).  On  a  vertical  microscope,  if  the  mirror  is  set  at  an  angle 
of  450,  the  axial  ray  will  be  at  right  angles  with  the  table  top  or  a  draw- 
ing board  which  is  horizontal,  and  a  drawing  made  under  these  condi- 
tion^ would  be  in  true  proportion  and  not  distorted.  The  stage  of  most 
microscopes,  however,  extends  out  so  far  at  the  sides  that  with  a  450 
mirror  the  image  appears  in  part  on  the  stage  of  the  microscope.  In 
order  to  avoid  this  the  mirror  may  be  depressed  to  some  point  below  45°, 
say  at  400  or  350  (Fig.  106-107).  But  as  the  axial  ray  from  the  mirror 
to  the  prism  must  still  be  reflected  horizontally,  it  follows  that  the  axial 
ray  will  no  longer  form  an  angle  of  90  degrees  with  the  drawing  sur- 
face, but  a  greater  angle.  If  the  mirror  is  depressed  to  350,  then  the 
axial  ray  must  take  an  angle  of  1  io°  with  a  horizontal  drawing  surface  ; 
see  the  geometrical  figure,  (Fig.  107).  To  make  the  angle  900  again, 
so  that  there  shall  be  no  distortion,  the  drawing  board  must  be  raised 
toward  the  microscope  200.  The  general  rule  is  to  raise  the  draw- 
ing board  twice  as  many  degrees  toward  the  microscope  as  the 
mirror  is  depressed  below  450.  Practically  the  field  for  drawing 
can  always  be  made  free  of  the  stage  of  the  microscope,  at  450,  at  400, 
or  at  350.  In  the  first  case  (450  mirror)  the  drawing  surface  should 
be  horizontal,  in  the  second  case  (400  mirror)  the  drawing  surface 
should  be  elevated  io°,  and  in  the  third  case  (350  mirror)  the  drawing 
board  should  be  elevated  200  toward  the  microscope.  Furthermore  it 
is  necessary  in  using  an  elevated  drawing  board  to  have  the  mirror  bar 
project  directly  laterally  so  that  the  edges  of  the  mirror  will  be  in 
planes  parallel  with  the  edges  of  the  drawing  board,  otherwise  there 
will  be  front  to  back  distortion,  although  the  elevation  of  the  drawing 
board  would  avoid  right  to  left  distortion.  If  one  has  a  micrometer 
ruled  in  squares  (?iet  ?nicrovieter)  the  distortion  produced  by  not  hav- 
ing the  axial  ray  at  right  angles  with  the  drawing  surface  may  be  very 
strikingly  shown.  For  example,  set  the  mirror  at  350  and  use  a  hori- 
zontal drawing  board.  With  a  pencil  make  dots  at  the  corners  of  some 
of  the  squares,  and  then  with  a  straight  edge  connect  the  dots.  The 
figures  will  be  considerably  longer  from  right  to  left  than  from  front  to 
back.  Circles  in  the  object  would  appear  as  ellipses  in  the  drawings, 
the  major  axis  being  from  right  to  left. 

The  angle  of  the  mirror  may  be  determined  with  a  protractor,  but 
that  is  troublesome.  It  is  much  more  satisfactory  to  have  a  quadrant 
attached  to  the  mirror  and  an  indicator  on  the  projecting  arm  of  the 
mirror.  If  the  quadrant  is  graduated  throughout  its  entire  extent,  or 
preferably  at  three  points,  450,  400  and  350,  one  can  set  the  mirror  at  a 
8 


ii4 


DRA  WING   WITH  THE  MICROSCOPE.  [CH    V. 

Fig.  108.  Fig.  107. 


Fig.  106. 


Fig  109. 


Figs.  106-109.  Abbe  Camera  Lucida  iti  position  to  avoid  distortion. 

Fig.  106.    The  Abbe  Camera  Lucida  with  the  mirror  at  33°. 

Axis,  Axis.    Axial  ray  from  the  microscope  and  from  the  drawing  surface. 

A  B.   Drawing  surface  raised  toward  the  microscope  20°. 

Foot.    The  foot  or  base  of  the  microscope. 

Mirror  with  quadrant  (Q).     The  mirror  is  seen  to  be  at  an  angle  of  33°. 

Ocular.    Ocular  of  the  microscope. 

P,  P.   Drawing  pencil,  and  the  cubical  prism  over  the  ocular. 

W.    Wedge  to  support  the  drawing  board. 

Fig.  107.  Geometrical  figure  of  the  preceding,  showing  the  angles  made  by  the 
axial  ray  with  the  mirror  and  the  necessary  elevation  of  the  drawing  board  to 
avoid  distortion.  From  the  equality  of  opposite  angles,  the  angle  of  the  axial  ray 
reflected  at  35°  must  make  an  angle  of  i/o°  with  a  horizontal  drawing  board.  The 
board  must  then  be  elevated  toward  the  microscope  20°  in  order  that  the  axial  ray 
may  be  perpendicular  to  it,  and  thus  fulfill  the  requirements  necessary  to  avoid  dis- 
tortion (§  170,  174). 

Fig.  108.  Upper  view  of  the  prism  of  the  camera  lucida.  A  considerable  portion 
of  the  face  of  the  prism  is  covered,  and  the  opening  in  the  silvered  surface  appears 
oval. 

Fig.  109.  Quadrant  to  be  attached  to  the  mirror  of  the  Abbe  Camera  Lucida  to 
indicate  the  angle  of  the  mirror.  As  the  angle  is  nearly  alzvays  at  45°,  400 ,  or  33°, 
only  those  angles  are  shown. 


en.  /'.] 


DRAWING  WITH  THE  MICROSCOPE. 


"5 


known  angle  in  a  moment,  then  the  drawing  hoard  can  he  hinged  and 
the  elevation  of  io°  and  200  determined  with  a  protractor.  The  draw- 
ing hoard  is  very  conveniently  held  up  by  a  broad  wedge.  Bv  marking 
the  position  of  the  wedge  for  io°  and  200  the  protractor  need  be  used 
but  once,  then  the  wedge  may  be  put  into  position  at  any  time  for  the 
proper  elevation. 

§  175.  Abbe  Camera  and  Inclined  Microscope. — It  is  ver}r  fatigu- 
ing to  draw  continuously  with  a  vertical  microscope,  and  many 
mounted  objects  admit  of  an  inclination  of  the  microscope,  when  one 
can  sit  and  work  in  a  more  comfortable  position.  The  Abbe  camera  is 
as  perfectly  adapted  to  use  with  an  inclined  as  with  a  vertical  micro- 
scope. All  that  is  requisite  is  to  be  sure  that  the  fundamental  law  is 
observed  regarding  the  axial  ray  of  the  image  and  the  drawing  surface, 
viz.,  that  they  should  be  at  right  angles.  This  is  very  easily  accom- 
plished as  follows  :  The  drawing  board  is  raised  toward  the  microscope 
twice  as  many  degrees  as  the  mirror  is  depressed  below  45 °  (§  174), 
then  it  is  raised  exactly  as  many  degrees  as  the  microscope  is  inclined, 
and  in  the  same  direction,  that  is,  so  the  end  of  the  drawing  board  shall 
be  in  a  plane  parallel  with  the  stage  of  the  microscope.  The  mirror 
must  have  its  edges  in  planes  parallel  with  the  edges  of  the  drawing 
board  also  (Fig.  no). 


Fig.  1 10.  Arrangement  of  the 
drawing  board  for  using  the  mi- 
croscope in  an  inclined  position 
with  the  Abbe  camera  lucida  {de- 
signed by  Mrs.  S.  P.  Gage). 


A  very  elaborate  and  convenient  drawing  board  has  been  devised  by 
Bernhard  (Zeit.  wiss.  Mikroskopie,  Vol.  XI,  (1894)  p.  298),  whereby 
the  proper  inclination  can  be  given  the  drawing  board  for  the  vertical 
microscope  and  also  for  an  inclined  microscope.  The  drawing  surface 
as  a  whole  can  be  raised  or  lowered  to  meet  the  needs  of  different  ob- 
jects. Fig.  1 1 1  shows  an  excellent  drawing  board  after  the  Bernhard 
form. 

§  176.  Drawing  with  the  Abbe  Camera  Lucida. — (A)  The  light 
from  the  microscope  and  from  the  drawing  surface  should  be  of  nearly 
equal  intensity,  so  that  the  image  and  the  drawing  pencil  can  be  seen 
with  about  equal  distinctness,.     This  may  be  accomplished  with  very 


n6 


DRAWING   WITH  THE  MICROSCOPE. 


\_CH.    V. 


low  powers  [6  mm.  and  lower  objectives)  by  covering  the  mirror  with 
white  paper  when  transparent  objects  are  to  be  drawn.  For  high  pow- 
ers it  is  best  to  use  a  substage  condenser.  Often  the  light  may  be  bal- 
anced by  using  a  larger  or  smaller  opening  in  the  diaphragm.  One 
can  tell  which  field  is  excessively  illuminated,  for  it  is  the  one  in  which 
objects  are  most  distinctly  seen.  If  it  is  the  microscopic,  then  the  image 
of  the  microscopic  object  is  very  distinct  and  the  pencil  is  invisible  or 
very  indistinct.  If  the  drawing  surface  is  too  brilliantly  lighted  the 
pencil  can  be  seen  clearly,  but  the  microscopic  image  will  be  very  ob- 
scure. 


Fig.  hi.  Drawing  Board  for  the  Abbe  Camera  Lucida  This  drawing  board, 
•  devised  by  the  Bausch  of  Lomb  Optical  Co.,  is  adjustable  vertically,  and  the  board 
may  be  inclined  to  prevent  distortion.  It  is  also  arranged  for  use  with  an  inclined 
microscope  by  having  the  base  board  hinged.  Microscope  and  drawing  surface 
are  then  inclined  together.  The  camera  lucida  has  a  graduated  arm  to  bear  the 
mirror  and  a  graduated  quadrant  at  the  mirror  joint  so  that  the  angle  of  the  mir- 
ror may  be  accurately  determined.  {See  also  Fig.  /ojk  (From  the  Bausch  & 
Lomb  Optical  Co.) 

When  opaque  objects,  that  is  objects  which  must  be  lighted  with  re- 
flected light  (§  59),  like  dark  colored  insects,  etc.,  are  to  be  drawn  the 
light  must  usually  be  concentrated  upon  the  object  in  some  way.     The 


C/f.   /'.]  DRAWING  Willi  THE  MICROSCOPl  117 

microscope  may  be  placed  in  a  very  stroiig  light  and  the  drawing  board 
shaded  or  the  light  may  be  concentrated  upon  the  object  by  means 

a  concave  mirror  or  a  hull's  eye  condenser  1  Fig.  g 

[f  the  drawing  surface  Is  too  brilliantly  illuminated,  it  may  be  sha 
by  placing  a  book  or  a  ground  glass  screen  between  it  and  the  window, 
also  by  putting  one  or  more  smoked  glasses  in  the  path  of  the  rays  from 
the  mirror  1  Fig.  [02  ( )  .  If  the  light  in  the  microscope  i--  too  intens 
it  may  he  lessened  by  using  white  paper  over  the  mirror,  or  hv  a 
ground  glass  screen  between  the  microscope  mirror  and  the  source  of 
light  (Piersol,  Amer.  M.  M.  Jour.,  1888,  p.  103).  It  is  also  an  excel- 
lent plan  to  blacken  the  end  of  the  drawing  pencil  with  carbon  ink. 
Sometimes  it  is  easier  to  draw  on  a  black  surface,  using  a  white  pencil 
or  style.  The  carbon  paper  used  in  manifolding  letters,  etc.,  may  be 
used,  or  ordinary  black  paper  may  be  lightly  rubbed  on  one  side  with  a 
moderately  soft  lead  pencil.  Place  the  black  paper  over  white  pamper 
and  trace  the  outlines  with  a  pointed  style  of  ivory  or  bone.  A  corre- 
sponding dark  line  will  appear  on  the  white  paper  beneath.  ( Jour. 
Roy.  Micr.  Soc,  1883,  p.  423). 

(A)  It  is  desirable  to  have  the  drawing  paper  fastened  with  thumb 
tacks,  or  in  some  other  way.  (B)  The  lines  made  while  using  the 
camera  lucida  should  be  very  light,  as  they  are  liable  to  be  irregular. 
(C)  Only  outlines  are  drawn  and  parts  located  with  a  camera  lucida. 
Details  are  put  in  free-hand.  (D)  It  is  sometimes  desirable  to  draw 
the  outline  of  an  object  with  a  moderate  power  and  add  the  details  with 
a  higher  power.  If  this  is  done  it  should  always  be  clearly  stated.  It 
is  advisable  to  do  this  only  with  objects  in  which  the  same  structure  is 
many  times  duplicated,  as  a  nerve  or  a  muscle.  In  such  an  object  all 
the  different  structures  could  be  shown,  and  by  omitting  some  of  the 
fibers  the  others  could  be  made  plainer  without  an  undesirable  enlarge- 
ment of  the  entire  figure. 

K)  If  a  drawing  of  a  given  size  is  desired  and  it  cannot  be  obtained 
by  any  combination  of  oculars,  objectives  and  lengths  of  the  tube  of 
the  microscope,  the  distance  between  the  camera  lucida  and  the  table 
may  be  increased  or  diminished  until  the  image  is  of  the  desired  size. 
This  distance  is  easily  changed  by  the  use  of  a  book  or  a  block,  but 
more  conveniently  if  one  has  a  drawine,  board  witli  adjustable  drawii 
surface  like  that  shown  in  Fig.  1 1 1.  The  image  of  a  few  spaces  of  the 
micrometer,  will  give  the  scale  of  enlargement,  or  the  power  may  be 
determined  for  the  special  case    >  [77-178 

P  )   It  is  of  the  greatest  advantage,  as  suggested  by  Eieinsius 
w.  Mikr.,  1889,  p.  367),  to  have  the  camera  lucida  hi:  that  the 


„8  DRAWING  WITH  THE  MICROSCOPE.  {CH.   V. 

prism  may  be  turned  off  the  ocular  for  a  moment's  glance  at  the  prepa- 
ration, and  then  returned  in  place  without  the  necessity  of  loosening 
screws  and  readjusting  the  camera.  This  form  is  now  made  by  several 
opticians,  and  the  quadrant  is  added  by  some.      Any  skilled  mechanic 

can  add  the  quadrant. 

8  177.  Magnification  of  the  Microscope  and  Size  of  Drawings 
with  the  Abbe  Camera  Lucida. — In  determining  the  standard  dis- 
tance of  250  millimeters  at  which  to  measure  the  image  in  getting  the 
magnification  of  the  microscope,  it  is  necessary  to  measure  from  the 
point  marked  P  on  the  prism  (Fig.  102;  to  the  axis  of  the  mirror  and 
then  vertically  to  the  drawing  board. 

In  getting  the  scale  to  which  a  drawing  is  enlarged  the  best  way  is 
to  remove  the  preparation  and  put  in  its  place  a  stage  micrometer,  and 
to  trace  a  few  (5  or  10)  of  its  lines  upon  one  corner  of  the  drawing. 
The  value  of  the  spaces  of  the  micrometer  being  given,  thus, 


1  Troth  mm. 

Fig.  112.  Showing  the  method  of  indicating  the  scale  at  which  a  drawing  zvas 
made. 

The  enlargement  of  the  figure  can  then  be  accurately  determined  at 
any  time  by  measuring  with  a  steel  scale  the  length  of  the  image  of  the 
micrometer  spaces  and  dividing  it  by  their  known  width. 

Thus,  suppose  the  5  spaces  of  the  scale  of  enlargement  given  with  a 
drawing  were  found  to  measure  25  millimeters  and  the  spaces  on  the 
micrometer  were  y^jth  millimeter,  then  the  enlargement  would  be 
25  -T-  jijj  =  500.  That  is,  the  image  was  drawn  at  a  magnification  of 
500  diameters. 

If  the  micrometer  scale  is  used  with  every  drawing,  there  is  no  need 
of  troubling  one's  self  about  the  exact  distance  at  which  the  drawing 
is  made,  convenience  may  settle  that,  as  the  special  magnification  in 
each  case  may  be  determined  from  the  scale  accompanying  the  picture. 
It  should  be  remembered,  however,  that  the  conditions  when  the  scale 
is  drawn  must  be  exactly  as  when  the  drawing  was  made. 

§  178.  Drawing  at  Slight  Magnification. — Some  objects  are  of 
considerable  size  and  for  drawings  should  be  enlarged  but  a  few  diame- 
ters,— 5  to  20.  By  using  sufficiently  low  objectives  and  different  ocu- 
lars a  great  range  may  be  obtained.  Frequently,  however,  the  range 
must  be  still  further  increased.  For  a  moderate  increase  in  size  the 
drawing  surface  may  be  put  farther  off,  or,  as  one  more  commonly 


CH   V.]  DRAWING  WITH  THE  MICROSCOPE. 

needs  less  rather  than  greater  magnification,  the  drawing  surface  may 
be  brought  nearer  the  mirror  of  the  camera  lucida  by  piling  books  or 
other  objects  on  the  drawing  board.  If  one  takes  the  precaution  to 
draw  a  scale  on  the  figure  under  the  same  conditions,  its  enlargement 

can  lie  readily  determined  (§  177  1. 

If  one  has  many  large  objects  to  draw  at  a  low  magnification,  then 
some  form  of  embryograph  is  very  convenient.  The  writer  has  made 
use  of  a  photographic  camera  and  different  photographic  objectives  for 
the  purpose.  The  object  is  illuminated  as  if  for  a  photograph  and  in 
place  of  the  ground  glass  a  plain  glass  is  used  and  on  this  some  tracing 
paper  is  stretched.  Nothing  is  then  easier  than  to  trace  the  outlines  of 
the  object.     See  also  Ch.  VIII. 

REFERENCES. 

Beale,  31,  355  ;  Behrens,  Kossel  and  Schiefferdecker,  77;  Carpenter-Dallii 
233  ;  Van  Heurck,  91;  American  Naturalist,  1886,  p.  1071,  1887,  pp.  [040  [043  ; 
Anier.  Monthly  Micr.  Jour.,  iSSS,  p.  103,  1890,  p.  94  ;  Jour.  Roy.  Micr.  Soc,  1881, 
p.  819,  1SS2,  p.  402,  1S83,  pp.  283,  560,  1S84,  p.  115,  18S6,  p.  516,  1888,  pp.  113,  809, 
79S  ;  Zeit.  wiss.  Mikroskopie,  1S84,  pp.  1-21,  1889,  p.  367.  1S93,  pp.  2S9-295. 
Here  is  described  an  excellent  apparatus  made  by  Winkel.  See  Zeiss'  catalog 
No  30,  and  the  15th  (1S96)  edition  of  the  Bausch  &  Lomb  Optical  Company  for 
improved  forms  of  the  Abbe  camera  lucida  and  for  improved  drawing  boards  to 
accompany  it. 


CHAPTER  VI. 


MICkn-SPI-CTROSCOPK  AND  POLARISCOPE. 


APPARATUS    AND    MATERIAL    REQUIRED    FOR   THIS    CHAPTER. 

Compound  microscope;  Micro-spectroscope  (?  179);  Watch-glasses  and  small 
vials,  slides  and  covers  {\  198)  ;  Various  substances  for  examination  (as  blood  and 
ammonium  sulphide,  permanganate  of  potash,  chlorophyll,  some  colored  fruit, 
etc.,  {$  199-209)  ;  Micro-polarizer  (§211)  ;  Selenite  plate  (g  220)  ;  Various  doubly 
refracting  objects,  as  crystals,  textile  fibers,  starch,  section  of  bone,  etc. 

MICRO-SPECTROSCOPE. 

I  179.  A  Micro-Spectroscope,  Spectroscopic  or  Spectral  Ocular,  is  a  direct  vision 
spectroscope  in  connection  with  a  microscopic  ocular.  The  one  devised  by  Abbe 
and  made  by  Zeiss  consists  of  a  direct  vision  spectroscope  prism  of  the  Amici  pat- 
tern, and  of  considerable  dispersion,  placed  over  the  ocular  of  the  microscope. 
This  direct  vision  or  Amici  prism  consists  of  a  single  triangular  prism  of  heavy  flint 
glass  in  the  middle  and  one  of  crown  glass  on  each  side,  the  edge  of  the  crown 
glass  prisms  pointing  toward  the  base  of  the  flint  glass  prism,  i.  e.,  the  edges  of  the 
crown  and  flint  glass  prisms  point  in  opposite  directions.  The  flint  glass  prism 
serves  to  give  the  dispersion  or  separation  into  colors,  while  the  crown  glass  prisms 
serve  to  make  the  emergent  rays  approximately  parallel  with  the  incident  rays,  so 
that  one  looks  directly  into  the  prism  along  the  axis  of  the  microscope. 

The  Amici  prism  is  in  a  special  tube  which  is  hinged  to  the  ocular  and  held  in 
position  by  a  spring.  It  may  be  swung  free  of  the  ocular.  In  connection  with 
the  ocular  is  the  slit  mechanism  and  a  prism  for  reflecting  horizontal  rays  verti- 
cally for  the  purpose  of  obtaining  a  comparison  spectrum  ($  192).  Finally  near 
the  top  is  a  lateral  tube  with  mirror  for  the  purpose  of  projecting  an  Angstrom 
scale  of  wave  lengths  upon  the  spectrum  (\  193,  Figs.  1 13-1 14). 

I  180.  Apparent  Reversal  of  the  Position  of  the  Colors  in  a  Direct  Vision  Spec- 
troscope.— In  accordance  with  the  statements  in  \  179  the  dispersion  or  separation 
into  colors  is  given  by  the  flint  glass  prism  or  prisms  and  in  accordance  with  the 
general  law  that  the  waves  of  shortest  length,  blue,  etc.,  will  be  bent  most,  conse- 
quently the  colors  have  the  position  indicated  in  the  top  of  Fig.  117,  also  above 
Fig.  118.  But  if  one  looks  into  the  direct  vision  spectroscope  or  holds  the  eye  close 
to  the  single  prism  (Fig.  118),  the  colors  will  appear  reversed  as  if  the  red  were 
more  bent.  The  explanation  of  this  is  shown  in  Fig.  118,  where  it  can  be  readily 
seen  that  if  the  eye  is  placed  at  E,  close  to  the  prism,  the  different  colored  rays 


CH.   17.]      MICRO  SPECTROSCOPE  AND  POLAR/SCOPE. 


I  2  I 


0  ^ 


Fig.  113.  Abbe's  Micto-  spectroscope.  Fig.  114. 

Longitudinal  Section  of  Slit  Mechanism  separately. 

the  whole  instrument.  [Plan  view,  Full  si 

{yi  Full  Size.) 

"  The  eye  lens  is  adjustable  so  as  to  accurately  focus  on  the  slit  situated  between 
the  lenses.  The  mechanism  for  contracting  and  expanding  the  slit  is  actuated 
the  screw  F  and  causes  the  laminae  to  move  symmetrically  \  Moz's  moveme 
The  slit  may  be  made  sufficiently  wide  so  as  to  include  the  whole  visual  field.  'J  he 
screw  H  serves  to  limit  the  length  of  the  slit  so  as  to  completely  fill  the  latter  with 
the  image  of  the  object  under  investigation  when  the  comparison  prism  is  inserted. 
The  comparison  prism  is  provided  with  a  lateral  fame  and  clips  to  hold  the  ob- 
ject and  the  illuminating  mirror.  .  III  these  parts  together  with  the  eye  pit .  c  are 
encased  in  a  drum. 

Above  the  eye-piece  is  placed  an  Amid  prism  of  great  dispersion  which  may  be 
turned  aside  about  the  pivot  A",  so  as  to  allow  of  the  adjustment  of  the  object  being 
controlled,  the  prism  being  retained  in  its  a.vial  position  by  the  spring  catch  L. 
A  scale  is  projected  on  the  spectrum  by  means  of  a  scale  tube  and  minor  attached 
to  the  prism  casing.  7 he  divisions  of  the  scale  indicate  in  decimals  of  a  micron 
the  wave  length  of  the  respective  section  of  the  spectrum.      Tin  to 

adjust  the  scale  relative  to  the  spectrum. 

The  instrument  is  inserted  in  the  tube  in  place  of  the  ordinary  eve  piece  and  is 
clamped  to  the  former  by  means  of  the  Si  1,  r,   .1/  in  such  a  position  that  the  <■ 
A  and  O,  which  respectively  serve  to  illuminate  the  comparison  prisms  and  the 
scale  of  wave  lengths,  are  simultaneously  illuminated."     (Zeiss  Catalog,  N  • 

will  appear  in  the  direction  from  which  they  reach  the  eye  ami  cons<  qu<  u 
crossed  in  being  projected  into  the  field  of  vision  and  the  real  position  ia  inverted. 
The  same  is  true  in  looking  into  the  micro  spectroscope.     The  a.  ; 
the  different  colors  may  be  determined  1>>  placing  some  ground  gl 
the  lens-paper  near  the  prism  and  observing  with  the  eye  at  thedistano 
vision.* 


*The  author  wishes  to  acknowledge  the  aid  rendered  by  Pro 

in  giving  the  explanation  offered  in  this  section. 


122 


MICRO  SPECTROSCOPE  AND  POL  A  R I  SCOPE.       [CH.    VI. 


Pcrmatx 

Fig.  i  15.  Various  Spectrums — All  except  that  of  Sodium  were  obtained  by  dif- 
fused day  light  with  the  slit  of  such  a  width  as  gave  the  most  distinct  Eraunhofer 
lines. 

It  frequently  occurs  that  with  a  substance  giving  several  absorption  bands  (e. 
g.,  chlorophyll)  the  density  or  thickness  of  the  solution  must  be  varied  to  show  all 
the  different  bands  clearly. 

Solar  Spectrum. —  With  diffused  day-light  and  a  narrow  slit  the  spectrum  is  not 
visible  much  beyond  the  fixed  li?ie  B.  In  order  to  extend  the  visible  spectrum  in 
the  red  to  the  line  A,  one  should  use  direct  sunlight  and  a  piece  of  ruby  glass  in 
place  of  the  watch  glass  in  Fig.  iij. 

Sodium  Spectrum.  —  The  line  spectrum  {\  1 82)  of  sodium  obtained  by  lighting 
the  microscope  with  an  alcohol  flame  in  which  some  salt  of  sodium  is  glowing. 
With  the  micro-spectroscope  the  sodium  line  seen  in  the  solar  spectrum  and  with 
the  incandescent  sodium  appears  single,  except  under  very  favorable  circumstances 
(2  193).  By  using  a  comparison  spectrum  of  day-light  with  the  sodium  spectrum 
the  light  and  dark  D-lines  zvill  be  seen  to  be  continuous  as  here  shozvn. 

Permanganate  of  Potash. —  This  spectrum  is  characterized  by  the  presence  of  five 
absorption  bands  in  the  middle  of  the  spectrum  and  is  best  shown  by  using  a  ^  per 
cent,  solution  of  permanganate  in  water  in  a  watch  glass  as  in  Fig.  nj. 

Met-hemoglobin. — The  absorptio?i  spectrum  of  met-hemoglobin  is  characterized 
by  a  considerable  darkening  of  the  blue  end  of  the  spectrum  and  of  four  absorp- 
tion hands,  one  in  the  red  near  the  line  C  and  two  between  D  and  E  nearly  in  the 
place  of  the  two  bands  of  oxy-hemoglobin  ;  finally  there  is  a  somewhat  faint,  wide 
band  near  F.  Such  a  met-hemoglobin  spectrum  is  best  obtained  by  making  a  solu- 
tion of  blood  in  water  of  such  a  concentration  that  the  izuo  oxy-hemoglobin  bands 
run  together  {\  202),  and  then  adding  three  or  four  drops  of  a  TV  per  cent,  aqueous 
solution  of  permanganate  of  potash  or  a  few  drops  of  hydrogen  dioxid  (H202). 
Soon  the  bright  red  zvill  change  to  a  brozunish  color,  when  it  may  be  examined. 

VARIOUS    KINDS    OF   SPECTRA. 


By  a  spectrum  is  meant  the  colored  bands  appearing  when  light  traverses  a  dis- 
persing prism  or  a  diffraction  grating,  or  is  affected  in  any  way  to  separate  the  dif- 
ferent wave  lengths  of  light  into  groups.  When  daylight  or  some  good  artificial 
light  is  thus  dispersed  one  gets  the  appearance  so  familiar  in  the  rainbow. 


CI  I,   VI, ]       MICRO-SPECTROSCOPE  AND  1 V  V. .  /  A'/.sv  \ >/  '/■/. 

\  181.   Continuous  Spectrum.  — In  case  a  good  artificial  light  or  the  I  :j^lit 

is  used  the  various  rainbow  <>r  spectral  colors  merge  gradually  into  one  another  in 
passing  from  end  to  end  of  the  spectrum.     There  are  no  breaks  01 

2.  1S2.   Line  Spectrum. — If  a  gas  is  made  incandescent,  tin-  spectrum  it  ; 
consists,  not  of  the  various  rainbow  colors,  hut  of  sharp,  narrow,  bright  lines,  the 
color  depending  on  the  substance.     All    tin-  rest  of  the   spectrum   isdaik.     These 
line  spectra  are  very  strikingly  shown  by  various  metals  heated  till  they  are  in  the 
form  of  incandescent  vapor. 

<;  [83.  Absorption  Spectrum.  —  By  this  is  meant  a  spectrum  in  which  then-  arc 
dark  lines  or  bands  in  the  spectrum.  The  most  striking  and  interesting  of  the  ab- 
sorption spectra  is  the  Solar  Spectrum,  or  spectrum  of  sunlight.  If  this  ra- 
ined carefully  it  will  be  found  to  be  crossed  by  dark  lines,  the  appearance  being  as 
if  one  were  to  draw  pen  marks  across  a  continuous  spectrum  at  various 
sometimes  apparently  between  the  colors  and  sometimes  in  the  midst  of  a  color. 
These  dark  lines  are  the  so  called  Fraunhofer  Lines.  Some  of  the  principal  ones 
have  been  lettered  with  Roman  capitals,  A,  B,  C,  D,  E,  F,  G,  II,  commencing  at 
the  red  end.  The  meaning  of  these  lines  was  for  a  long  time  enigmatical,  but  it 
is  now  known  that  they  correspond  with  the  bright  lines  of  a  line  spectrum 
For  example,  if  sodium  is  put  in  the  flame  of  a  spirit  lamp  it  will  vaporize  and  be- 
come luminous.  If  this  light  is  examined  there  will  be  seen  one  or  two  bright 
yellow  bands  corresponding  in  position  with  D  of  the  solar  spectrum  1  Fig.  i  I 
If  now  the  spirit  lamp-flame,  colored  by  the  incandescent  sodium,  is  placed  in  the 
path  of  the  electric  light,  and  it  is  examined  as  before,  there  will  be  a  continuous 
spectrum,  except  for  dark  lines  in  place  of  the  bright  sodium  lines.  That  is,  the 
comparatively  cool  yellow  light  of  the  spirit  lamp  cuts  off  or  absorbs  the  intensely 
hot  yellow  light  of  the  electric  light  ;  and  although  the  spirit  flame  sends  a  yellow- 
light  to  the  spectroscope  it  is  so  faint  in  comparison  with  the  electric  light  that  the 
sodium  lines  appear  dark.  It  is  believed  that  in  the  sun's  atmosphere  there  are 
incandescent  metal  vapors  (sodium,  iron,  etc.),  but  that  they  are  so  cool  iu  com- 
parison with  the  rays  of  their  wave  length  in  the  sun  that  the  cooler  light  of  the 
incandescent  metallic  vapors  absorb  the  light  of  corresponding  wave  length,  ami 
are,  like  the  spirit  lamp  flame,  unable  to  make  up  the  loss,  and  therefore  the  pres- 
ence of  the  dark  lines. 

\  184.  Absorption  Spectra  from  Colored  Substances. — While  the  solar  spectrum 
is  an  absorption  spectrum,  the  term  is  more  commonly  applied  to  the  spectra  ob- 
tained with  light  which  has  passed  through  or  has  been  reflected  from  colored  ob- 
jects which  are  not  self-luminous. 

It  is  the  special  purpose  of  the  micro-spectroscope  to  investigate  the  spectra  "t 
colored  objects  which  are  not  self-luminous,  as  blood   and  other   liquids,  ' 
minerals,  as  monazite,  etc.     The  spectra  obtained  by  examining  the  light  refla 
from   these  colored  bodies  or  transmitted   through   them,  possess,  like   the   solar 
spectrum   dark   lines  or  bands,  but   the  bands   are   usually   much   wider  and 
sharply  defined.      Their  number  and  position  depend  on  the  Bubsl 
stitution   (Fig.   116),    and   their  width,  in  part,    115011   the   thickness   oi    thi 
With  some  colored  bodies,  no  definite  ban. Is  are  present     The  spectrun  |  ly 

restricted  at  one  or  both   ends  and    various  of  the  other  colors  an    •     D       •:  Vi.lv 
lessened  in  intensity.     This  is  true  of  many  colored  truits 

\  185.   Angstrom  and  Stokes"  Law  of  Absorption  Spectra.     The  wave  lengtJ 
light  absorbed  by  a  body   when  light  is  transmitted  through  BOm 


124 


MICRO-SPECTROSCOPE  AND  POLARISCOPE.       \CH.   VI. 


A     a     B    C 


.90      SO 


70 


u 


1 1 11 


<L    <Oc.Jagsfodlo'^  g 


FlG.  i  16.  Absorption  Spectrum  of  Oxy-Hemoglobin  or  arterial  blood  (/)  and  of 
Hemoglobin  or  venous  blood  (2).     {From  Gamgee  and  McMunn.) 

.  I,  />',  C,  D,  E,  E,  G,  H.  Some  of  the  Principal  Fraunhofer  lines  of  the  solar 
spectrum  {I  J  83). 

.go,  .80,  .70.  .60,  .50,  .40.  Wave  lengths  in  microtis,  as  shown  in  Angstrom's 
scale  {\  193).  It  will  be  seen  that  the  wave  lengths  increase  toward  the  red  and 
decrease  toward  the  violet  end  of  the  spectrum. 

Red,  Orange,  Yellow,  etc.  Color  regions  of  the  spectrum.  Indigo  should  come 
between  the  blue  and  the  violet  to  complete  the  seven  colors  usually  given.  It  was 
omitted  through  inadvertence. 

are  precisely  the  waves  radiated  from  it  when  it  becomes  self  luminous.  For  ex- 
ample, a  piece  of  glass  that  is  yellow  when  cool,  gives  out  blue  light  when  it  is  hot 
enough  to  be  self-luminous.  Sodium  vapor  absorbs  two  bauds  of  yellow  light  (D 
lines)  ;  but  when  light  is  not  sent  through  it,  but  itself  is  luminous  and  examined 
as  a  source  of  light  its  spectrum  gives  bright  sodium  lines,  all  the  rest  of  the  spec- 
trum being  dark. 

\  186.  Law  of  Color. — The  light  reaching  the  eye  from  a  colored,  solid,  liquid 
or  gaseous  body  lighted  with  white  light,  will  be  that  due  to  white  light  less  the 
.  light  waves  that  have  been  absorbed  by  the  colored  body.  Or  in  other  words,  it 
will  be  due  to  the  wave  lengths  of  light  that  finally  reach  the  eye  from  the  object. 
For  example,  a  thin  layer  of  blood  under  the  microscope  will  appear  yellowish 
green,  but  a  thick  layer  will  appear  pure  red.  If  now  these  two  layers  are  exam- 
ined with  a  micro-spectroscope,  the  thin  layer  will  show  all  the  colors,  but  the  red 
end  will  be  slightly,  and  the  blue  end  considerably  restricted,  and  some  of  the  colors 
will  appear  of  considerably  lessened  intensity.  Finally  there  may  appear  two 
shadow-like  bands,  or  if  the  layer  is  thick  enough,  two  well-defined  dark  bands  in 
the  green  ($  202). 

If  the  thick  layer  is  examined  in  the  same  way,  the  spectrum  will  show  only  red 
with  a  little  orange  light,  all  the  rest  being  absorbed.  Thus  the  spectroscope  shows 
which  colors  remain,  in  part  or  wholly,  and  it  is  the  mixture  of  this  remaining  or 
un absorbed  light  that  gives  color  to  the  object. 

\  187.  Complementary  Spectra. — While  it  is  believed  that  Angstrom's  law  (|  185) 
is  correct,  there  are  many  bodies  on  which  it  cannot  be  tested,  as  they  change  in 
chemical  or  molecular  constitution  before  reaching  a  sufficiently  high  temperature 
to  become  luminous.  There  are  compounds,  however,  like  those  of  didvmium, 
erbium  and  terbium,  which  do  not  change  with  the  heat  necessary  to  render  them 
luminous,  and  with  them  the  incandescence  and  absorption  spectra  are  mutually 
complementary,  the  one  presenting  bright  lines  where  the  other  presents  dark 
ones  (Daiiiell). 


CII.   /'/.]      MICRO-SPECTROSCOPE  AND  POLARISCOPE.  125 

ADJUSTING   THE    MICRO  SPECTROSCOPB. 

£  [88.  The  micro-spectroscope,  or  spectroscopic  ocular,  is  put  in  the 
place  of  the  ordinary  ocular  in  the  microscope,  and  clamped  to  the  top  of 
the  tube  by  means  of  a  screw  for  the  purpose. 

£  [89.  Adjustment  of  the  Slit. — -In  place  of  the  ordinary  diaphragm! 
with  circular  opening,  the  spectral  ocular  has  a  diaphragm  comp 
two  movable  knife  edges  by  which  a  slit  like  opening  of  greater  01 
width  and  length  may  be  obtained  at  will  by  the  use  of  screws  for  the 
purpose.     To  adjust  the  slit,  depress  the  lever  holding  the  prism-tube 
iu  position  over  the  ocular,  and  swing  the  prism  aside.     <  me  can  then 
look  into  the  ocular.     The  lateral  screw  should  be  used  and  the  knife 
edges  approach  till  they  appear  about  half  a  millimeter  apart.     It   now 
the  Amici  prism  is  put  back  in  place  and  the  microscope  well  lighted, 
one  will  see  a  spectrum  by  looking  into  the  upper  end  of  the  spe< 
scope.     If  the  slit  is  too  wide,  the  colors  will  overlap  in   the  middl 
the  spectrum  and  be  pure  only  at  the  red  and  blue  ends  ;  and  the  Fraun- 
hofer  or  other  bands  in  the  spectrum  will  be  faint  or  invisible.      Dust  on 
the  edges  of  the  slit  gives  the  appearance  of  longitudinal  streaks  on  the 
spectrum. 

s,  191).  Mutual  Arrangement  of  Slit  and  Prism. — In  order  that 
the  spectrum  may  appear  as  if  made  up  of  colored  bands  going  directly 
across  the  long  axis  of  the  spectrum,  the  slit  must  be  parallel  with  the 
refracting  edge  of  the  prism.  If  the  slit  and  prism  are  not  thus  mutu- 
ally arranged,  the  colored  bands  will  appear  oblique,  and  the  whole 
spectrum  may  be  greatly  narrowed.  If  the  colored  bands  are  oblique, 
grasp  the  prism  tube  and  slowly  rotate  it  to  the  right  or  to  the  left  until 
the  various  colored  bands  extend  directly  across  the  spectrum. 

vj  191.  Focusing  the  Slit. — In  order  that  the  lines  or  bauds  in  the 
spectrum  shall  be  sharply  defined,  the  eye-lens  of  the  ocular  should  be 
accurately  focused  on  the  slit.  The  eye-lens  is  movable,  and  when  the 
prism  is  swung  aside  it  is  very  easy  to  focus  the  slit  as  one  focused  for 
the  ocular  micrometer  (^  161  ).  If  one  now  uses  daylight  there  will  be 
seen  in  the  spectrum  the  dark  Frauuhofer  lines  (  Fig.  1 1"  E.  F., 

To  show  the  necessity  of  focusing  the  slit,  move  the  eye  1<  ns  down 
or  up  as  far  as  possible,    and    the    Frauuhofer   lines   cannot    i 
While  looking  into  the  spectroscope  move  the  ocular  lens  up  01  down, 
and  when  it  is  focused  the  Frauuhofer  lines  will  reappear.     As  the  dif- 
ferent colors  of  the  spectrum  have  different  wave  lengths,  it  is  111 
to  focus  the  slit  for  each  color  if  the  sharpest  possible  pictui 

It  will  be  found  that  the  eye-lens  of  the  ocular  must 


126 


MICRO-SPECTROSCOPE  AND  POLAR/SCOPE.       {CH.    VI. 


I  S  ti-1 


Fig.  117. 


Fig.  118. 


Fig.  119. 


Fig.  117  (1).  Sectiofi  of  the  tube  and  stage  of  the  microscope  with  the  spectral 
ocular  or  micro  spectroscope  in  position. 

Amici  Prism  (g  167).  —  The  direct  vision  prism  of  Ami ci  in  which  the  central 
shaded  prism  of  flint  glass  gives  the  dispersion  or  separation  into  colors,  while  the 
end  prisms  of  crown  glass  cause  the  rays  to  emerge  approximately  parallel  ivith 
the  axis  of  the  microscope.  A  single  ray  is  represented  as  entering  the  prism  and 
this  is  divided  into  three  groups  [Red,  Yellow,  Blue),  which  emerge  from  the 


CH.  VI]      MICRO-SPECTROSCOPE  AND  POLAR/SCOPE,  u; 

prism,  the  red  being  least  and  the  blue  >>/  tsi  bent  toward  the  base  of  the  flint  prism 
(see  Fig.   M.S'. 

Hinge.— The  hinge  on  which  the  prism  tube  turns  when  it  is  swung  off  the 

ocular. 

Ocular  {\  179) — The  ocular  in  which  the  slit  mechanism  takes  the  place  0/ the 
diaphragm  \\  189).  The  eye  lens  is  movable  as  in  a  micrometer  ocular,  that 
the  slit  may  be  accurately  focused for  tin-  different  colors  |  \  191  . 

S.  Screw  for  setting  the  scale  of  wave  lengths  1  \  193). 

S' .  Screw  for  regulating  the  width  of  the  slit  \\  1S9). 

S,f.  Screw  for  clamping  the  micro -spectroscope  to  the  tube  of  the  ».     ■        pe. 

Scale  Tube.  —  The  tube  near  the  upper  end  containing  the  Angstrom    cale  and 
the  lenses  for  projecting  the  image  upon  the  upper  face  of  the  Amici  prism,  whet 
it  is  reflected  upward  to  the  eye  with  the  different  colored  rays.     At  the  right  is  a 
special  mirror  for  lighting  the  scale.     For  arrang  ing  and  focusing  the  stale,  |  see 
I  193)- 

Slit.  —The  linear  opening  between  the  knife  edges  Through  the  slit  the  light 
passes  to  the  prism.  It  must  be  arranged  parallel  zuith  the  refracting  edge  of  the 
prism,  and  of  such  a  width  that  the  Fraunhofer  or  Fixed  Lines  arc  very  clearly 
and  sharply  defined  when  the  eye-lens  is  properly  focused  {\  1S9-191  1. 

Stage.  —  The  stage  of  the  microscope.  This  supports  a  watch-glass  with  sloping 
sides  for  containing  the  colored  liquid  to  be  examined. 

(3;  Comparison  Prism  with  tube  for  colored  liquid  (C.  L. ),  and  mirror.      The 
prism  rejlects  horizontal  rays  vertically,  so  that  when  the  prism  is  made  t. 
part  of  the  slit  tzuo  parallel  spectra  may  be  seen,  one  from   light  sent  directly 
through  the  entire  microscope  and  one  from  the  light  reflected  upward  from  the 
comparison  prism. 

(4)  View  of  the  Slit  Mechanism  from  below. — Slit,  the  linear  space  between  the 
knife  edges  through  which  the  light  passes. 

P.  Comparison  prism  beneath  the  slit  and  covering  part  of  it  at  will. 

S.  S' .  Screws  for  regulating  the  -width  and  length  of  the  slit. 

Fig.  1 18.  Flint-Glass  Prism  showing  the  separation  or  dispersion  of  white  . 
into  the  three  groups  of  colored  rays  {Red,  Yellow,  Blue  |,  the  blue  rays  being  bent 
the  most  from  the  refracting  edge  (§  1S0). 

Fig.  1 19.  Sectional  View  of  a  Microscope  with  the  Polariscope  in  Position  1  \  209- 
217). 

Analyzer  and  Polarizer. —  They  are  represented  with  corresponding  .■;•.//- 

lei  so  that  the  polarized  beam  could  traverse  freely  the  analyzer.     If  eiti;.  < 
were  rotated  900  they  would  be  crossed  and  no  light  would  traverse  the  at 
unless  some  polarizing  substance  were  used  as  object  \\  212). 
alyzer  tube  so  that  the  analyzer  may  be  raised  or  lowered  to  adjust  ilfot 
of  level  of  the  eye-point  in  different  oculat  \    '..  21  |. 

Pointer  and  Scale.  —  The  pointer  attached  to  the  analyzer  and  the 
circle  clamped  {by  the  screw  S)  to  the  tube  of  the  microscope.     The  p 
scale  enable  one  to  determine  the  exact  amount  of  rotation  of  the  ana 

Object. —  The  object  whose  character  is  to  be  investigated  by  /  g  M. 


r28  .VfCRO-SPECTROSCOPE  AND  POLARISCOPE.       [Cf/.IV. 

the  slit  for  the  sharpest  focus  of  the  red  end  than  for  the  sharpest  focus 
of  the  lines  at  the  blue  end.  This  is  because  the  wave  length  of  red  is 
markedly  greater  than  for  blue  light. 

Longitudinal  dark  lines  on  the  spectrum  may  be  due  to  irregularity 
of  the  edge  of  the  slit  or  to  the  presence  of  dust.  They  are  most 
troublesome  with  a  very  narrow  slit. 

c<  [92.  Comparison  or  Double  Spectrum. — In  order  to  compare 
the  spectra  of  two  different  substances  it  is  desirable  to  be  able  to  exam- 
ine their  spectra  side  by  side.  This  is  provided  for  in  the  better  forms 
of  micro-spectroscopes  by  a  prism  just  below  the  slit,  so  placed  that  the 
light  entering  it  from  a  mirror  at  the  side  of  the  drum  shall  be  totally 
reflected  in  a  vertical  direction,  and  thus  parallel  with  the  rays  from  the 
microscope.  The  two  spectra  will  be  side  by  side  with  a  narrow  dark 
line  separating  them.  If  now  the  slit  is  well  focused  and  daylight  be 
sent  through  the  microscope  and  into  the  side  to  the  reflecting  or  com- 
parison prism,  the  colored  bands  and  the  Fraunhofer  dark  lines  will 
appear  directly  continuous  across  the  two  spectra.  The  prism  for  the 
comparison  spectrum  is  movable  and  may  be  thrown  entirely  out  of  the 
field  if  desired.  When  it  is  to  be  used,  it  is  moved  about  half  way 
across  the  field  so  that  the  two  spectrums  shall  have  about  the  same 
width. 

£  193.   Scale  of  Wave  Lengths. — In  the  Abbe  micro-spectroscope 
the  scale  is  in  a  separate  tube  near  the  top  of  the  prism  and  at  right 
angles  to  the  prism-tube.     A  special  mirror  serves  to  light  the  scale, 
which  is  projected  upon  the  spectrum  by  a  lens  in  the  scale-tube.     This 
scale  is  of  the  Angstrom  form,  and  the  wave  lengths  of  any  part  of  the 
spectrum  may  be  read  off  directly,  after  the  scale  is  once   set  in  the 
proper  position,  that  is,  when  it  is  set  so  that  any  given  wave  length 
on  the  scale  is  opposite  the  part  of  the  spectrum  known  by  previous 
investigation  to  have  that  particular  wave  length.     The  point  most  often 
selected  for  setting  the  scale  is  opposite  the  sodium  lines  where  the  wave 
length  is,  according  to  Angstrom,  0.5892  jx.     In  adjusting  the  scale,  one 
may  focus  very  sharply  the  dark  sodium  line  of  the  solar  spectrum  and 
set  the  scale  so  that  the  number  0.589  is  opposite  the  sodium  or  D  line, 
or  a  method  that  is  frequently  used  and  serves  to  illustrate  §  171,  is  to 
sprinkle  some  salt  of  sodium  (carbonate  of  sodium  is  good)  in  an  alco- 
hol lamp  flame  and  to  examine  this  flame.      If  this  is  done  in  a  dark- 
ened place  with   a  spectroscope,   a   narrow  bright   band   will   be   seen 
in  the    yellow    part    of    the   spectrum.     If    now    ordinary   daylight  is 
sent  through    the   comparison    prism,    the    bright  line   of  the  sodium 
will  be  seen  to  be  directly  continuous  with   the  dark  line  at  D  in  the 


CH.   VI.]      MICRO-SPECTROSCOPE  AND  POLARISCOPE.  120 

s  »lar  sp  ictrum  (  Fig.  1 14).     Now,  by  reflecting  light  into  the  seal 

the  image  of  the  scale  will  appear  on  the  spectrum,  and  by  a  screw  just 

under  the  scale-tube,   hut  in  the  prism-tube,  the  proper  point  on  the 

smle  Co. 589 /a)  can  be  brought  opposite  the  sodium  hind.  All  the  scale 
will  then  give  the  wave  lengths  directly.  Sometimes  the  scale  is  oblique 
to  the  spectrum.  This  may  he  remedied  by  turning  the  prism-tub  ■ 
slightly  one  way  or  the  other.  It  may  he  due  to  the  wrong  position  of 
the  scale  itself.  If  so,  grasp  the  milled  ring  at  the  distal  end  of  the 
scale-tube  and,  while  looking  into  the  spectroscope,  rotate  the  tube  until 
the  lines  of  the  scale  are  parallel  with  the  Fraunhofer  lines.  It  is  neces- 
sary in  adjusting  the  scale  to  he  sure  that  the  larger  number,  0.70,  is  at 
the  red  end  of  the  spectrum. 

The  numbers  on  the  scale  should  be  very  clearly  defined.  If  they  do 
not  so  appear,  the  scale-tube  must  be  focused  by  grasping  the  outer  tube 
of  the  scale-tube  and  moving  it  toward  or  from  the  prism-tube  until  the 
scale  is  distinct.  In  focusing  the  scale,  grasp  the  outer  scale-tube  with 
one  hand  and  the  prism-tube  with  the  other,  and  push  or  pull  in  oppo- 
site directions.  In  this  way  one  will  be  less  liable  to  injure  the  spec- 
troscope. 

ij  194.  Designation  of  Wave  Length. — Wave  lengths  of  light  are 
designated  by  the  Greek  letter  A,  followed  by  the  number  indicating  the 
wave  length  in  some  fraction  of  a  meter.  With  the  Abbe  micro-spec- 
troscope the  micron  is  taken  as  the  unit  as  with  other  microscopical 
measurements  (§  157).  Various  units  are  in  use,  as  the  one  hundred 
thousandth  of  a  millimeter,  millionths  or  ten  millionths  of  a  millimeter. 
If  these  smaller  units  are  taken,  the  wave  lengths  will  be  indicated 
either  as  a  decimal  fraction  of  a  millimeter  or  as  whole  numbers.  Thus, 
according  to  Angstrom,  the  wave  length  of  sodium  light  is  5892  ten 
millionths  mm.,  or  589.2  millionths,  or  58.92  one  hundred  thousandths, 
or  0.5892  one  thousandth  mm.,  or  0.5892  /a.  The  last  would  be  indi- 
cated thus,  AD  =  0.5892 /a. 

ij  195.  Lighting  for  the  Micro-speclroscope. — For  opaque  objects 
a  strong  light  should  be  thrown  on  them  either  with  a  concave  mirror 
or  a  condensing  lens.  For  transparent  objects  the  amount  of  the  sub- 
stance and  the  depth  of  color  must  be  considered.  As  a  general  rule  it 
is  well  to  use  plenty  of  light,  as  that  from  an  Abbe  illuminator  with  a 
large  opening  in  the  diaphragm,  or  with  the  diaphragm  entirely  removed. 
For  very  small  objects  and  thin  layers  of  liquids  it  may  be  better  to  use 
less  light.  One  must  try  both  methods  in  a  given  case,  and  learn  by 
experience. 

The  direct  and  the  comparison  spectrums  should  be  about  equallv 

9 


130  M ICRO-SPECTROSCOPE  AND  POLARISCOPE.       \CH.   VI. 

illuminated.  One  can  manage  this  by  putting  the  object  requiring  the 
greater  amount  of  illumination  on  the  stage  of  the  microscope  and  light- 
ing it  with  the  Abbe  illuminator.  In  lighting  it  is  found  in  general 
that  for  red  or  yellow  objects,  lamp-light  gives  very  satisfactory  results. 
Fbr  the  examination  of  blood  and  blood  crystals,  the  light  from  a  petro- 
leum lamp  is  excellent  ($201-203).  For  objects  with  much  blue  or 
violet,  daylight  or  artificial  light  rich  in  blue  light  is  best.  The  new 
acetylene  light  ought  to  be  very  satisfactory  (§  65). 

Furthermore,  one  should  be  on  his  guard  against  confusing  the  ordin- 
ary absorption  bands  with  the  Fraunhofer  lines  when  daylight  is  used. 
With  lamp-light  the  Fraunhofer  lines  are  absent  and,  therefore,  not  a 
source  of  possible  confusion. 

§  196.  Objectives  to  Use  with  the  Micro-spectroscope. — If  the 
material  is  of  considerable  bulk,  a  low  objective  ( 18  to  50  mm. )  is  to  be 
preferred.  This  depends  on  the  nature  of  the  object  under  examina- 
tion, however.  In  case  of  individual  crystals  one  should  use  sufficient 
magnification  to  make  the  real  image  of  the  crystal  entirely  fill  the 
width  of  the  slit.  The  length  of  the  slit  may  then  be  regulated  by  the 
screw  on  the  side  of  the  drum,  and  also  by  the  comparison  prism.  If 
the  object  does  not  fill  the  whole  slit  the  white  light  entering  the  spec- 
troscope with  the  light  from  the  object  might  obscure  the  absorption 
bands. 

In  using  high  objectives  with  the  micro-spectroscope  one  must  very 
carefully  regulate  the  light  (§  58,  102),  and  sometimes  shade  the  object. 

^  197.  Focusing  the  Objective. — For  focusing  the  objective  the 
prism-tube  is  swung  aside,  and  then  the  slit  made  wide  by  turning  the 
adjusting  screw  at  the  side.  When  the  slit  is  open,  one  can  see  objects 
when  the  microscope  is  focused  as  with  an  ordinary  ocular.  After  an 
object  is  focused,  it  may  be  put  exactly  in  position  to  fill  the  slit  of  the 
spectroscope,  then  the  knife  edges  are  brought  together  till  the  slit  is  of 
the  right  width  ;  if  the  slit  is  then  too  long  it  may  be  shortened  by  using 
one  of  the  mechanism  screws  on  the  side,  or  if  that  is  not  sufficient,  by 
bringing  the  comparison  prism  farther  over  the  field.  If  one  now 
replaces  the  Amici  prism  and  looks  into  the  microscope,  the  spectrum  is 
liable  to  have  longitudinal  shimmering  lines.  To  get  rid  of  these  focus 
up  or  down  a  little  so  that  the  microscope  will  be  slightly  out  of  focus. 

§  198.  Amount  of  Material  Necessary  for  Absorption  Spectra 
and  its  Proper  Manipulation. — The  amount  of  material  necessary  to 
give  an  absorption  spectrum  varies  greatly  with  different  substances, 
and  can  be  determined  only  by  trial.  If  a  transparent  solid  is  under 
investigation  it  is  well  to  have  it  in  the  form  of  a  wedge,  then  succes- 


CH.   /'/.]      MICRO-SPECTROSCOPE  AND  POLAR/SCOPE.  [31 

sive  thicknesses  can  be  brought  under  the  microscope.  If  a  liquid  sub- 
stance is  being  examined,  a  watch  glass  with  sloping  sides  forms  an 
excellent  vessel  to  contain  it,  then  successive  thicknesses  of  the  liquid 
can  be  brought  into  the  field  as  with  the  wedge-shaped  solid.  1 
quently  only  a  very  weak  solution  is  obtainable  ;  in  this  case  it  can  be 
placed  in  a  homoeopathic  vial,  or  in  some  glass  tubing  sealed  at  the  end, 
then  one  can  look  lengthwise  through  the  liquid  and  get  the  effect  of  a 
more  concentrated  solution.  For  minute  bodies  like  crystals  or  blood 
corpuscles,  one  may  proceed  as  described  in  the  previous  section. 

MICRO-SPECTROSCOPE — EXPERIMENTS. 

§  199.  Put  the  micro-spectroscope  in  position,  arrange  the  slit  and 
the  Amici  prism  so  that  the  spectrum  will  show  the  various  spectral 
colors  going  directly  across  it  (§  188-189)  and  carefully  focus  the  slit. 
This  may  be  done  either  by  swinging  the  prism-tube  aside  and  proceed- 
ing as  for  the  ocular  micrometer  (§  161),  or  by  moving  the  eye-lens  of 
the  ocular  up  and  down  while  looking  into  the  micro-spectroscope  until 
the  dark  lines  of  the  solar  spectrum  are  distinct.  If  they  cannot  be 
made  distinct  by  focusing  the  slit,  then  the  light  is  too  feeble  or  the  slit 
is  too  wide  (§  191).  With  the  lever  move  the  comparison  prism  across 
half  the  field  so  that  the  two  spectra  shall  be  of  about  equal  width.  For 
lighting,  see  §195. 

§  200.  Absorption  Spectrum  of  Permanganate  of  Potash. — Make 
a  solution  of  permanganate  of  potash  in  water  of  such  a  strength  that 
a  stratum  3  or  4  mm.  thick  is  transparent.  Put  this  solution  in  a  watch- 
glass  with  sloping  sides,  and  put  it  under  the  microscope.  Use  a  50  mm. 
or  16  mm.  objective,  and  use  the  full  opening  of  the  illuminator.  Light 
strongly.  Look  into  the  spectroscope  and  slowly  move  the  watch-glass 
into  the  field.  Note  carefully  the  appearance  with  the  thin  stratum  of 
liquid  at  the  edge  and  then  as  it  gradually  thickens  on  moving  the 
watch-glass  still  farther  along.  Count  the  absorption  bauds  and  note 
particularly  the  red  and  blue  ends.  Compare  carefully  with  the  com- 
parison spectrum  (Fig.  113).     For  strength  of  .solution  see  §  202. 

§201.  Absorption  Spectrum  of  Blood. — Obtain  blood  from  a 
recently  killed  animal,  or  flame  a  needle,  and  after  it  is  cool  prick  the 
finger  two  or  three  times  in  a  small  area,  then  wind  a  handkerchief  or  a 
rubber  tube  around  the  base  of  the  finger,  and  squeeze  the  finger  with 
the  other  hand.  Some  blood  will  ooze  out  of  the  pricks.  Rinse  this  off 
in  a  watch-glass  partly  filled  with  water.  Continue  to  add  the  blood 
until  the  water  is  quite  red.     Place  the  watch-glass  of  diluted  blood  un 


1*2  MICRO  SPECTROSCOPE  AND  POLARISCOPE.       [CH.   VI. 


:> 


der  the  microscope  in  place  of  the  permanganate,  using  the  same  object- 
ive, etc.  Note  carefully  the  spectrum.  It  would  be  advantageous  to 
determine  the  wave  length  opposite  the  center  of  the  dark  bands.  This 
in  iy  I-  ■  done  easily  by  setting  the  scale  properly  as  described  in  §  193. 
M  ike  another  preparation,  but  use  a  homoeopathic  vial  instead  of  a 
watch-glass.  Cork  the  vial  and  lay  it  down  upon  the  stage  of  the  mi- 
croscope. Observe  the  spectrum.  It  will  be  like  that  in  the  watch- 
glass.  Remove  the  cork  and  look  through  the  whole  length  of  the  vial. 
The  bands  will  be  very  much  darker,  and  if  the  solution  is  thick  enough 
only  red  and  a  little  orange  will  appear.  Re-insert  the  cork  and  incline 
the  vial  so  that  the  light  traverses  a  very  thin  layer,  then  gradually  ele- 
vate the  vial  and  the  effect  of  a  thicker  and  thicker  layer  may  be  seen. 
Note  especially  that  the  two  characteristic  bands  unite  and  form  one 
wide  band  as  the  stratum  of  liquid  thickens.  Compare  with  the  fol- 
lowing : 

Add  to  the  vial  of  diluted  blood  a  drop  or  two  of  ammonium  sulphide, 
such  as  is  used  for  a  reducing  agent  in  chemical  laboratories.  Shake 
the  bottle  gently  and  then  allow  it  to  stand  for  ten  or  fifteen  minutes. 
Examine  it  and  the  two  bands  will  have  been  replaced  by  a  single,  less 
clearly  defined  band  in  about  the  same  position.  The  blood  will  also 
appear  somewhat  purple.  Shake  the  vial  vigorously  and  the  color  will 
change  to  the  bright  red  of  fresh  blood.  Examine  it  again  with  the  spec- 
troscope and  the  two  bauds  will  be  visible.  After  five  or  ten  minutes 
another  examination  will  show  but  a  single  band.  Incline  the  bottle  so 
that  a  very  thin  stratum  may  be  examined.  Note  that  the  stratum  of 
liquid  must  be  considerably  thicker  to  show  the  absorption  band  than 
was  necessary  to  show  the  two  bands  in  the  first  experiment.  Further- 
more, while  the  single  band  may  be  made  quite  black  on  thickening  the 
stratum,  it  will  not  separate  into  two  bauds  with  a  thinner  stratum.  In 
this  experiment  it  is  very  instructive  to  have  a  second  vial  of  fresh  dilut- 
ed blood,  say  that  from  the  watch-glass,  before  the  opening  of  the  com- 
parison prism.  The  two  banded  spectrum  will  then  be  in  position  to 
be  compared  with  the  spectrum  of  the  blood  treated  with  the  ammonium 
sulphide. 

The  two  banded  spectrum  is  of  oxy- hemoglobin ,  or  arterial  blood,  the 
single  banded  spectrum  is  of  hemoglobin  (sometimes  called  reduced 
hemoglobin)  or  venous  blood,  that  is,  the  respiratory  oxygen  is  present 
in  the  two  banded  spectrum  but  absent  from  the  single  banded  spectrum. 
When  the  bottle  was  shaken  the  hemoglobin  took  up  oxygen  from  the 
air  and  became  oxy-hemoglobin,  as  occurs  in  the  lungs,  but  soon  the 
ammonium  sulphide  took  away  the  respiratory  oxygen,  thus  reducing 


CI  I,    VI,}       MICRO-SPECTROSCOPE  AND  POLAR ISCOPE.  [33 

the  oxy-hemoglobin  to  hemoglobin.     This  may  be  rep  -a  ted  many  tin; 
I  Fig.  114). 

^  202.  Met-Hemoglobin. — The  absorption  spectrum  of  met-hemo- 
globin  is  characterized  by  a  considerable  darkening  of  the  blue  end 
the  spectrum  and  of  four  absorption  bands,  one  in  the  red  near  the  line 
C  and  two  between  I)  and  E,  nearly  in  the  place  of  the  two  bands  "t 
oxy-hemoglobin  ;  finally  there  is  a  somewhat  faint,  wide  band  near  F. 
Such  a  met-hemoglobin  spectrum  is  best  obtained  by  making  a  solution 
of  blood  in  water  of  such  a  concentration  that  the  two  oxy -hemoglobin 
bands  run  together  (§201),  and  then  adding  three  or  four  drops  of  a 
y1^  per  cent,  aqueous  solution  of  permanganate  of  potash.  Soon  the 
bright  red  will  change  to  a  brownish  color,  when  it  may  be  examined 
(Fig.  113). 

£  203.  Carbon  Monoxide  Hemoglobin  (CO  Hemoglobin). — To 
obtain  this  one  may  kill  an  animal,  after  auaesthetization,  in  illuminat- 
ing gas,  or  one  may  allow  illuminating  gas  to  bubble  through  some 
blood  already  taken  from  the  body.  The  gas  should  bubble  through  a 
minute  or  two.  The  oxygen  will  be  displaced  by  carbon  monoxide. 
This  forms  quite  a  stable  compound  with  hemoglobin,  and  is  of  a  bright 
cherry-red  color.  Its  spectrum  is  nearly  like  that  of  oxy-hemoglobin, 
but  the  bands  are  farther  toward  the  blue.  Add  several  drops  of  am- 
monium sulphide  and  allow  the  blood  to  stand  some  time.  No  reduc- 
tion will  take  place,  thus  forming  a  marked  contrast  to  solutions  of  oxy- 
hemoglobin. By  the  addition  of  a  few  drops  of  glacial  acetic  acid  a 
dark  brownish  red  color  is  produced. 

§  204.  Carmine  Solution. — Make  a  solution  of  carmine  by  putting 
TVth  gram  of  carmine  in  100  cc.  of  water  and  adding  10  drops  of  strong 
ammonia.  Put  some  of  this  in  a  watch-glass  or  in  a  small  vial  and  com- 
pare the  spectrum  with  that  of  oxy-hemoglobin  or  carbon  monoxide  he- 
moglobin. It  has  two  bands  nearly  in  the  same  position,  thus  giving 
the  spectrum  a  striking  similarity  to  blood.  If  now  several  drops,  15 
or  20,  of  glacial  acetic  acid  are  added  to  the  carmine,  the  bands  remain 
and  the  color  is  not  very  markedly  changed,  while  with  either  oxy-hemo- 
globin or  CO-hemoglobiu  the  color  would  be  very  markedly  changed 
from  the  bright  red  to  a  dull  reddish  brown,  and  the  spectrum,  if  any 
could  be  seen,  would  be  markedly  different.  Carmine  and  <  )  hemoglo- 
bin can  be  distinguished  by  the  use  of  ammonium  sulphide,  the  carmine 
remaining  practically  unchanged  while  the  blood  shows  the  single  band 
of  hemoglobin  ($  201).  The  acetic  acid  serves  to  differentiate  the  CO- 
hemoglobin  as  well  as  the  O-hemoglobiu. 

i$  jos.    Colored  Bodies  not  giving  Distinctly  Banded  Absorp- 


134  MICRO-SPECTROSCOPE  AND  POLARISCOPE.       [CH.   VI. 

tion  Spectra. — Some  quite  brilliantly  colored  objects,  like  the  skin  of 
a  red  apple,  do  not  give  a  banded  spectrum.  Take  the  skin  of  a  red 
apple,  mount  it  on  a  slide,  put  on  a  cover-lass  and  add  a  drop  of  water 
at  the  edge  of  the  cover.  Put  the  preparation  under  the  microscope 
and  observe  the  spectrum.  Although  no  bands  will  appear,  in  some 
cases  at  least,  yet  the  ends  of  the  spectrum  will  be  restricted  and  vari- 
ous regions  of  the  spectrum  will  not  be  so  bright  as  the  comparison 
spectrum.  Here  the  red  color  arises  from  the  mixture  of  the  uuab- 
sorbed  wave  lengths,  as  occurs  with  other  colored  objects.  In  this 
case,  however,  not  all  the  light  of  a  given  wave  length  is  absorbed, 
consequently  there  are  no  clearly  defined  dark  bands,  the  light  is  simply 
less  brilliant  in  certain  regions  and  the  red  rays  so  predominate  that 
they  give  the  prevailing  color. 

^  206.  Nearly  Colorless  Bodies  with  Clearly  Marked  Absorp- 
tion Spectra. — In  contradistinction  to  the  brightly  colored  objects  with 
no  distinct  absorption  bands  are  those  nearly  colorless  bodies  and  solu- 
tions which  give  as  sharply  defined  absorption  bands  as  could  be  de- 
sired. The  best  examples  of  this  are  afforded  by  solutions  of  the  rare 
earths,  didymium,  etc.  These  in  solutions  that  give  hardly  a  trace  of 
color  to  the  eye  give  absorption  bands  that  almost  rival  the  Fraunhofer 
lines  in  sharpness. 

§  207.  Absorption  Specftra  of  Minerals. — As  example  take  some 
monazite  sand  on  a  slide  and  either  mount  it  in  balsam  (see  Ch.  VII), 
or  cover  and  add  a  drop  of  water.  The  examination  may  be  made  also 
with  the  dry  sand,  but  it  is  less  satisfactory.  Light  well  with  trans- 
mitted light,  and  move  the  preparation  slowly  around.  Absorption 
bands  will  appear  occasionally.  Swing  the  prism-tube  off  the  ocular, 
open  the  slit  and  focus  the  sand.  Get  the  image  of  one  or  more  grains 
directly  in  the  slit,  then  narrow  and  shorten  the  slit  so  that  no  light 
can  reach  the  spectroscope  that  has  not  traversed  the  grain  of  sand. 
The  spectrum  will  be  very  satisfactory  under  such  conditions.  It  is 
frequently  of  great  service  in  determining  the  character  of  unknown 
mineral  sands  to  compare  their  spectra  with  known  minerals.  If  the 
absorption  bands  are  identical,  it  is  strong  evidence  in  favor  of  the 
identity  of  the  minerals.     For  proper  lighting  see  S  195- 

§  208.  While  the  study  of  absorption  spectra  gives  one  a  great  deal 
of  accurate  information,  great  caution  must  be  exercised  in  drawing 
conclusions  as  to  the  identity  or  even  the  close  relationship  of  bodies 
giving  approximately  the  same  absorption  spectra.  The  rule  followed 
by  the  best  workers  is  to  have  a  known  body  as  control  and  to  treat  the 
unknown  body  and  the  known  body  with  the  same  reagents,  and  to 


CH.   VI.]      MICRO-SPECTROSCOPE  AND  POLARISCOPE.  135 

dissolve  them  in  the  same  medium.  If  all  the  reactions  are  identical 
then  the  presumption  is  very  strong  that  the  bodies  arc  identical  or 
very  closely  related.  For  example,  while  one  might  be  in  doubt  be- 
tween a  solution  of  oxy-  or  CO-hemoglobin  and  carmine,  the  addition 
of  ammonium  sulphide  would  serve  to  change  the  double  to  a  single 
band  in  the  Ohemoglobin,  and  glacial  acetic  acid  would  enable  one  to 
distinguish  between  the  CO-blood  and  the  carmine,  although  the  am- 
monium sulphide  would  not  enable  one  to  make  the  distinction. 
Furthermore  it  is  unsafe  to  compare  objects  dissolved  in  different 
media.  The  same  objects  as  "cyanine  and  aniline  blue  dissolved  in 
alcohol  give  a  very  similar  spectrum,  but  in  water  a  totally  different 
one."  "  Totally  different  bodies  show  absorption  bands  in  exactly  the 
same  position  (solid  nitrate  of  uranium  and  permanganate  of  potash  in 
the  blue)."  (MacMunn).  The  rule  given  by  MacMunn  is  a  good 
one  :  "  The  recognition  of  a  body  becomes  more  certain  if  its  spectrum 
consists  of  several  absorption  bands,  but  even  the  coincidence  of  these 
bands  with  those  of  another  body,  is  not  sufficient  to  enable  us  to  infer 
chemical  identity  ;  what  enables  us  to  do  so  with  certainty  is  the  fact : 
that  the  two  solutions  give  bands  of  equal  intensities  in  the  same  parts  of 
the  spectrum  which  undergo  analogous  changes  o?i  the  addition  of  the 
same  reagent.' ' 

REKKREXCES  TO  THE  MICRO-SPECTROSCOPE   AND   SPECTRUM   ANALYSIS. 

The  micro-spectroscope  is  playing  an  ever  increasingly  important  role  in  the 
spectrum  analysis  of  animal  and  vegetable  pigments,  and  of  colored  mineral  and 
chemical  substances,  therefore  a  somewhat  extended  reference  to  literature  will  be 
given.  Full  titles  of  the  books  and  periodicals  will  be  found  in  the  Bibliography 
at  the  end. 

Angstrom,  Recherches  sur  le  spectre  solaire,  etc.  Also  various  papers  in  period- 
icals. See  Royal  Soc's  Cat'l  Scientific  Papers  ;  Anthony  &  Rrackett  ;  Beale,  p. 
269  ;  Behrens,  p.  139  ;  Kossel  und  Schiefferdecker,  p.  63  ;  Carpenter,  p.  104  ;  Brown- 
ing, How  to  Work  with  the  Spectroscope,  and  in  Monthly  Micr.  Jour.,  II,  p.  65  ; 
Daniell,  Principles  of  Physics.  The  general  principles  of  spectrum  analysis  are 
especially  well  stated  in  this  work,  pp.  435-455  ;  Davis,  p.  342  ;  Dippel,  p.  277  ; 
Frey  ;  Gamgee,  p.  91  ;  Halliburton  ;  Hogg,  p.  122  ;  also  in  Monthly  Micr.  Jour., 
Vol.  II,  on  colors  of  flowers  ;  Jour.  Roy.  Micr.  Soc.,  1880,  1SS3,  and  in  various  other 
vols.  ;  Kraus  ;  Lockyer  ;  M'Kendrick  ;  MacMunn  ;  and  also  in  Philos.  Trans.  R.  S., 
1886;  various  vols,  of  Jonr.  Physiol.;  Ncigeli  und  Schwendener  ;  Proctor;  Ref. 
Hand-Book  Med.  Sciences,  Vol.  I,  p.  577,  VI,  p.  516,  VII,  p.  426  ;  Roscoe  ;  Schel- 
len  ;  Sorby,  in  Beale,  p.  269;  also  Proc.  R.  S.,  1874,  p.  31,  1N67,  p.  4.33  ;  see  also 
in  the  Scientific  Review,  Vol.  V,  p.  66,  Vol.  II,  p.  419.  The  larger  works  011  Ph\  bi- 
ology, Chemistry  and  Physics  may  also  be  consulted  with  profit. 

Vogel,  Spectrum  analysis,  also  in  Nature,  Vol.  xix,  p.  .495,  on  absorption  spectra. 
The  bibliography  in  MacMunn  is  excellent  and  extended. 


136  MICRO-SPECTROSCOPE  AND  POLARISCOPE.       {CH.   VI. 

M ICRO-POLARISCOPE. 

\  209.  Ttie  niicro-polariscope,  or  polarizer,  is  a  polariscope  used  in  connection 

with  a  microscope. 

The  most  common  and  typical  form  consists  of  two  Nicol  prisms,  that  is,  two 
somewhat  elongated  rhombs  of  Iceland  spar  cut  diagonally  and  cemented  together 
with  Canada  balsam.  These  Nicol  prisms  are  then  mounted  in  such  a  way  that 
the  light  passes  through  them  lengthwise,  and  in  passing  is  divided  into  two  rays 
of  plane  polarized  light.  The  one  of  these  rays  obeying  most  nearly  the  ordinary 
law  of  refraction  is  called  the  ordinary  ray,  the  one  departing  farthest  from  the 
law  is  called  the  extra-ordinary  ray.  These  two  rays  are  not  only  polarized,  but 
polarized  in  planes  almost  exactly  at  right  angles  to  each  other.  The  Nicol  prism 
totally  reflects  the  ordinary  ray  at  the  cemented  surface  as  it  meets  that  surface  at 
an  angle  greater  than  the  critical  angle,  and  only  the  extraordinary  or  less  refracted 
ray  is  transmitted. 

\  210.  Polarizer  and  Analyzer.— The  polarizer  is  one  of  the  Nicol  prisms.  It  is 
placed  beneath  the  object  and  in  this  way  the  object  is  illuminated  with  polarized 
light.  The  analyzer  is  the  other  Nicol  and  is  placed  at  some  level  above  the  object, 
very  conveniently  above  the  ocular. 

When  the  corresponding  faces  of  the  polarizer  and  analyzer  are  parallel  i.  <?., 
when  the  faces  through  which  the  oblique  section  passed  are  parallel,  light  passes 
freely  through  the  analyzer  to  the  eye.  If  these  corresponding  faces  are  at  right 
angles,  that  is,  if  the  Nicols  are  crossed,  then  the  light  is  entirely  cut  off  and  the 
two  transparent  prisms  become  opaque  to  ordinary  light.  There  are  then,  in  the 
complete  revolution  of  the  analyzer,  two  points,  at  0°  and  180°,  where  the  corre- 
sponding faces  are  parallel  and  where  light  freely  traverses  the  analyzer.  There 
are  also  two  crossing  points  of  the  Nicols,  at  900  and  2700,  where  the  light  is  extin- 
guished.    In  the  intermediate  points  there  is  a  sort  of  twilight. 

\  211.  Putting  the  Polarizer  and  Analyzer  in  Position. — Swing  the  diaphragm 
carrier  of  the  Abbe  illuminator  out  from  under  the  illuminator,  remove  the  disk 
diaphragm  or  open  widely  the  iris  diaphragm  and  place  the  analyzer  in  the  dia- 
phragm carrier,  then  swing  it  back  under  the  illuminator.  Remove  the  ocular, 
put  the  graduated  ring  on  the  top  of  the  tube  and  then  replace  the  ocular  and  put 
the  analyzer  over  the  ocular  and  ring.  Arrange  the  graduated  ring  so  that  the  indi- 
cator shall  stand  at  o°  when  the  field  is  lightest.  This  may  be  done  by  turning  the 
tube  down  so  that  the  objective  is  near  the  illuminator,  then  shading  the  stage  so 
that  none  but  polarized  light  shall  enter  the  microscope.  Rotate  the  analyzer  until 
the  lightest  possible  point  is  found,  then  rotate  the  graduated  ring  till  the  index 
stands  at  o°.  The  ring  may  then  be  clamped  to  the  tube  by  the  side  screw  for  the 
purpose.  Or,  more  easily,  one  may  set  the  index  at  o°,  clamp  the  ring  to  the 
microscope,  then  rotate  the  draw-tube  of  the  microscope  till  the  field  is  lightest. 

£  212.  Adjustment  of  the  Analyzer. — The  analyzer  should  be  capable  of  moving 
up  and  down  in  its  mounting,  so  that  it  can  be  adjusted  to  the  eye-point  of  the  ocu- 
lar with  which  it  is  used.  If  on  looking  into  the  analyzer  with  parallel  Nicols  the 
edge  of  the  field  is  not  sharp,  or  if  it  is  colored,  the  analyzer  is  not  in  a  proper  posi- 
tion with  reference  to  the  eye-point,  and  should  be  raised  or  lowered  till  the  edge 
of  the  field  is  perfectly  sharp  and  as  free  from  color  as  the  ocular  with  the  analyzer 
removed. 

\  213.   Objectives  to  Use  with  the  Polariscope. — Objectives  of  the  lowest  power 


CH.  VI.]      MICRO-SPECTROSCOPE  AND  POLAR/SCOPE. 

may  be  used,  and  also  all  intermediate  forms  up  to  a  2  mm.  homogeneous  immer- 
sion. Still  higher  objectives  may  be  used  if  desired.  In  general,  however,  the 
lower  powers  are  somewhat  more  satisfactory.  A  good  rule  to  follow  in  this  i  ase 
is  the  general  rule  in  all  microscopic  work, — use  the  power  that  most  clearly  and 
satisfactorily  shows  the  object  under  investigation. 

§214.  Lighting  for  Micro-Polariscope  Work. —  Follow  the  general  directions 
given  in  Chapter  II.  It  is  especially  necessary  to  shade  the  object  so  that  no  1111- 
polarized  light  can  enter  the  objective,  otherwise  the  field  cannot  be  sufficiently 
darkened.  No  diaphragm  is  used  over  the  polarizer  for  most  examinations.  1  >i  n 
sunlight  may  be  used  to  advantage  with  some  objects,  and  as  a  rule  the  objeel 
would  best  be  very  transparent. 

§215.  Mounting  Objects  for  the  Polariscope. — So  far  as  possible  objects  should 
be  mounted  in  balsam  to  render  them  very  transparent.  In  many  cases  objects 
mounted  in  water  do  not  give  satisfactory  polariscopic  appearances.  For  example, 
if  starch  is  mounted  dry  or  in  water,  the  appearances  are  not  so  striking  as  in  a 
balsam  mount  (Davis,  p.  337  ;  Suffolk). 

\  216.  Purpose  of  a  Micro-Polariscope. — The  object  of  a  micro-polariscope  is  to 
determine,  in  microscopic  masses,  one  or  more  of  the  following  points  :  i.\ 
Whether  the  body  is  singly  refractive,  mono-refringent,  or  isotropic,  that  is,  opti- 
cally homogeneous,  as  are  glass  and  crystals  belonging  to  the  cubical  system  ;  (B) 
Whether  the  object  is  doubly  refractive  or  anisotropic,  uniaxial  or  biaxial  ;  (C) 
Pleochroism  ;  (D)  The  rotation  of  the  plane  of  polarization,  as  with  solutions  of 
sugar,  etc.  ;  (E)  To  aid  in  petrology  and  mineralogy  ;  (F)  To  aid  in  the  determi- 
nation of  very  minute  quantities  of  crystallizable  substances  ;  (G)  For  the  produc- 
tion of  colors. 

For  petrological  and  mineral ogical  investigations  the  microscope  should  possess 
a  graduated,  rotating  stage  so  that  the  object  can  be  rotated,  and  the  exact  angle 
of  rotation  determined.  It  is  also  found  of  advantage  in  investigating  objects  witli 
polarized  light  where  colors  appear,  to  combine  a  polariscopic  and  spectroscope 
(Spectro-Polariscope). 

MICRO-POLARISCOPE — EXPERI-MKXTS. 

§  217.  Arrange  the  polarizer  and  analyzer  as  directed  above  ($  21 1) 
and  use  a  16  mm.  objective  except  when  otherwise  directed. 

(A)  Isotropic  or  Singly  Refractive  Objects. — Light  the  micro- 
scope well  and  cross  the  Xicols,  shade  the  stage  and  make  the  field  as 
dark  as  possible  (§  210).  As  an  isotropic  substance,  put  an  ordinary 
glass  slide  under  the  microscope.  The  field  will  remain  dark.  As  an 
example  of  a  crystal  belonging  to  the  cubical  system  and  hence  i- 
tropic,  make  a  strong  solution  of  common  salt  (sodium  chloride  Xa  CI.  . 
put  a  drop  on  a  slide  and  allow  it  to  crystallize,  put  it  under  the  micro- 
scope, remove  the  analyzer,  focus  the  crystals  and  then  replace  the  an- 
alyzer and  cross  the  Nicols.  The  field  and  the  crystals  will  remain 
dark. 

(B)  Anisotropic  or  Doubly  Refracting  Objects. —  Make  a  f; 


138  MICRO-SPECTROSCOPE  AND  POLARISCOPE.       [CH.   VI. 

preparation  of  carbonate  of  lime  crystals  like  that  described  for  pedesis 
(§  142),  or  use  a  preparation  in  which  the  crystals  have  dried  to  the 
slide,  use  a  5  or  3  mm.  objective,  shade  the  object  well,  remove  the  an- 
alyzer and  focus  the  crystals,  then  replace  the  analyzer.  Cross  the 
Nicols.  In  the  dark  field  will  be  seen  multitudes  of  shining  crystals, 
and  if  the  preparation  is  a  fresh  one  in  water,  part  of  the  smaller  crys- 
tals will  alternately  flash  and  disappear.  By  observing  carefully,  some 
of  the  larger  crystals  will  be  found  to  remain  dark  with  crossed  Nicols, 
others  will  shine  continuously.  If  the  crystals  are  in  such  a  position 
that  the  light  passes  through  them  parallel  with  the  optic  axis,*  the 
crystals  are  isotropic  like  the  salt  crystal  and  remain  dark.  If,  how- 
ever, the  light  traverses  them  in  any  other  direction  the  ray  from  the 
polarizer  is  divided  into  two  constituents  vibrating  in  planes  at  right 
angles  to  each  other,  and  one  o'f  these  will  traverse  the  analyzer,  hence 
such  crystals  will  appear  as  if  self-luminous  in  a  dark  field.  The  experi- 
ment with  these  crystals  from  the  frog  succeeds  well  with  a  2  mm.  ho- 
mogeneous immersion. 

As  further  illustration  of  anisotropic  objects,  mount  some  cotton 
fibers  in  balsam  (Ch.  VII),  also  some  of  the  lens  paper  (§  107).  These 
furnish  excellent  examples  of  vegetable  fibers. 

Striated  muscular  fibers  are  also  very  well  adapted  for  polarizing  ob- 
jects. 

As  examples  of  biaxial  crystals,  allow  some  borax  solution  to  dry 
and  crystallize  on  a  slide  ;  use  the  crystals  as  object.  As  all  doubly  re- 
fracting objects  restore  the  light  with  crossed  Nicols,  they  are  some- 
times called  depolarizing. 

(C)  Pleochroism. — This  is  the  exhibition  of  different  tints  as  the  an- 
alyzer is  rotated.  An  excellent  subject  for  this  will  be  found  in  blood 
crystals. 

(D)  For  the  aid  given  by  the  polariscope  in  micro-chemistry,  see 
(Ch.  VII). 

(E)  See  works  on  petrology  and  mineralogy  for  the  application  of 
the  micro-polarizer  in  those  subjects. 

^  218.  Production  of  Colors. — For  the  production  of  gorgeous 
colors,  a  plate  of  selenite  giving  blue  and  yellow  colors  is  placed  between 

*The  optic  axis  of  doubly  refracting  crystals  is  the  axis  along  which  the  crystal 
is  not  doubly  refracting,  but  isotropic  like  glass.  When  there  is  but  one  such 
axis,  the  crystal  is  said  to  be  uniaxial,  if  there  are  two  such  axes  the  crystal  is 
said  to  be  bi-axial. 

The  crystals  of  carbonate  of  lime  from  the  frog  (see  \  142)  are  uniaxial  crystals. 
Borax  crystals  are  bi-axial. 


CH.   VI]      MICRO-SPECTROSCOPE  AND  POLARISCOPE.  i.v, 

the  polarizer  and  the  object.  If  properly  mounted,  the  selenite  is  very 
conveniently  placed  on  the  diaphragm  carrier  of  the  Abbe  illuminator, 
just  above  the  polarizer.     A  thin  plate  or  film  of  mica  also  answers  well. 

It  is  not  necessary  to  use  a  selenite  or  piece  of  mica  for  the  produc- 
tion of  the  most  glorious  colors  in  many  objects.  One  of  the  most 
beautiful  preparations,  and  one  of  the  most  instructive  also,  may  be 
prepared  as  follows  :  Heat  some  xylene  balsam  on  a  slide  until  the 
xylene  is  nearly  evaporated.  Add  some  crystals  of  the  hypnotic  medi- 
cine, sulphonal  and  warm  till  the  sulphonal  is  melted  and  mixes  with 
the  balsam.  While  the  balsam  is  still  melted  put  on  a  cover-glass.  If 
one  gets  perfect  crystals  there  will  be  shown  not  only  most  beautiful 
colors,  but  the  black  cross  with  perfection.      (Clark  I. 

It  is  very  instructive  and  interesting  to  examine  organic  and  inor- 
ganic substances  with  a  micro-polarizer.  *If  the  objects  enumerated  in 
^  144  were  all  examined  with  polarized  light  an  additional  means  of  de- 
tecting them  would  be  found. 

REFERENCES    TO    THE    POLARISCOPE    AND    TO    THE    USE  OF   POLARIZED 

LIGHT. 

Anthony  &  Brackett ;  Behrens,  133  ;  Behrens,  Kossel  trad  Schiefferdecker  ;  Car- 
noy,  61  ;  Carpenter- Dallinger,  262,  269,  992  ;  Clark  ;  Daniell,  494  ;  Davis  ;  v.  Ebe- 
ner  ;  Gage;  Gamgee  ;  Halliburton,  36,  272;  Hogg,  133,  729;  Lehmann  ;  M'Ken- 
drick  ;  Nageli  und  Schweudener,  299;  Queckett  ;  Suffolk,  125;  Valentin.  Physi- 
cal Review,  I.,  p.  127.     Daniell,  Physics  for  Medical  Students. 


CHAPTER  VII. 


SLIDES  AND  COVER-GLASSES;  MOUNTING;  ISOLATION, 
SECTIONING  BY  THE  COLLODION  AND  THE  PARAF- 
FIN METHODS  ;  LABELING  AND  STORING  MICRO- 
SCOPICAL PREPARATIONS  ;  EXPERIMENTS  IN  MICRO- 
CHEMISTRY. 


APPARATUS    AND    MATERIAL   FOR    THIS    CHAPTER. 

Microscope,  compound  and  simple  (Ch.  I)  ;  Micro-Spectroscope  and  polariscope 
(Ch.  VI);  vSlides  and  cover-glasses  (#219-220)  ;  Cleaning  mixtures  for  glass  (^227)  ; 
Alcohol  and  distilled  or  filtered  water  (g  222)  ;  fine  forceps  for  handling  cover- 
glasses  (§  222-226)  ;  Old  handkerchiefs  or  lens  paper  (g  107,  223).  Paper  boxes  for 
storing  cover-glasses  ($  223,  225)  ;  Cover-glass  measurer  (Figs.  120-122)  ;  Mount- 
ing material, — Farraut's  solution,  glycerin,  glycerin-jelly  and  Canada  balsam  (\  243, 
246)  ;  Centering  card  and  card  for  serial  sections  (g  236)  ;  Material  for  dissociation 
and  for  the  paraffin  and  collodion  method  ($  244)  ;  Material  for  paraffin  and  collo- 
dion sectioning  ($  250)  ;  Net-micrometer  for  arranging  minute  objects  like  diatoms 
($  3*7)  !  Labels  {\  309)  ;  Carbon  ink  for  writing  labels  (§  295)  ;  Writing  diamond 
(§  295)  ;  Shellac  cement  (g  316)  ;  Cabinet  (§  296)  ;  Re-agents  for  experiments  in 
micro-chemistry  (#315). 

SEIDES   AND    COVER-GEASSES. 

§  219.  Slides,  Glass  Slides  or  Slips,  Microscopic  Slides  or  Slips. 
These  are  strips  of  clear  flat  glass  upon  which  microscopic  specimens 
are  usually  mounted  for  preservation  and  ready  examination.  The  size 
that  has  been  almost  universally  adopted  for  ordinary  preparations  is  25 
x  76  millimeters  (1x3  inches).  For  rock  sections,  slides  25  x  45  mm. 
or  32  x  32  mm.  are  used  ;  for  serial  sections,  slides  25  x  76  mm. ,  50  x  75 
mm.  or  37  x  87  mm.  are  used.  For  special  purposes,  slides  of  the  nec- 
essary size  are  employed  without  regard  to  any  conventional  standard. 

Whatever  size  of  slide  is  used,  it  should  be  made  of  clear  glass  and 
the  edges  should  be  ground.  It  is  altogether  false  economy  to  mount 
microscopic  objects  on  slides  with  ungrouud  edges.  It  is  unsafe  also  as 
the  ungrouud  edges  are  liable  to  wound  the  hands. 

§  220.  Cleaning  Slides. — For  new  slides  a  thorough  rinsing  in  clean 
water  with  subsequent  wiping  with  a  soft  towel,  and  then  an  old  soft 


ClI.   VII]  SLIDES  AND  COVER-GLASSES,  141 

handkerchief,  usually  fits  them  for  ordinary  use.     If  th  not  satis- 

factorily cleaned  in  this  way,  soak  them  a  short  time  in  50$    or  75$ 

alcohol,  let  them  drain  for  a  few  moments  on  a  clean  towel  <>r  on  blot- 
ting paper,  and  then  wipe  with  a  soft  cloth.  In  handling  the  slides 
grasp  them  by  their  edges  to  avoid  soiling  the  face  of  the  slide.  After 
the  slides  are  cleaned  they  should  be  stored  in  a  place  as  free  a^  possible 
from  dust. 

For  used  slides,  if  only  water,  glycerin  or  glycerin  jelly  has  been  11 
on  them,  they  may  be  cleaned  with  water,  or  preferably,  warm  water 
and  then  with  alcohol  if  necessary.  Where  balsam,  or  any  oil}-  or  gum- 
my substance  has  been  used  upon  the  slides,  they  may  be  freed  from  tin- 
balsam,  etc.,  by  soaking  them  for  a  week  or  more  in  one  of  the  clean- 
ing mixtures  for  glass.  If  they  are  first  soaked  in  xylene,  benzin  or  tur- 
pentine to  dissolve  the  balsam,  then  soaked  in  the  cleaning  mixture,  the 
time  required  will  be  much  shortened  ($  227).  After  all  foreign  mat- 
ter is  removed  the  slides  should  b?  very  thoroughly  rinsed  in  water  to 
remove  all  the  cleaning  mixture.  They  may  then  be  treated  as  directed 
for  new  slides. 

If  slides  with  large  covers,  as  in  mounted  series,  are  put  into  the 
cleaning  mixture,  the  swelling  of  the  balsam  is  liable  to  break  the  covers. 
Dissolving  away  the  balsam  with  turpentine,  etc.,  avoids  this,  and 
greatly  shortens  the  time  necessary  for  cleaning  the  old  slides  and  covers. 

Another  excellent  method  for  balsam  mounts  is  to  heat  the  slides  until 
the  balsam  is  soft  and  then  remove  the  cover-glasses.  The  cleaning 
mixture  can  then  act  on  the  entire  surface.  It  should  be  said,  however, 
that  at  the  present  price  of  slides  and  cover-glasses  it  is  hardly  worth 
while  to  clean  those  that  have  been  used  in  balsam  mounting. 

§  221.  Cover-Glasses  or  Covering  Glasses. —These  are  circular 
or  quadrangular  pieces  of  thin  glass  used  for  covering  and  protecting 
microscopic  objects.  They  should  be  very  thin,  /,,",,  to  1-„',,l  millimeter 
(see  table,  §  27).  It  is  better  never  to  use  a  cover-glass  over  ,'-'„",,  mm. 
thick,  then  the  preparation  may  be  studied  with  a  2  mm.  oil  immersion 
as  well  as  with  lower  objectives.  Except  for  objects  wholly  unsuited 
for  high  powers,  it  is  a  great  mistake  to  use  cover-glasses  thicker  than 
the  working  distance  of  a  homogeneous  objective  (£47).  Indeed,  if 
one  wishes  to  employ  high  powers,  the  thicker  the  sections  the  thinner 
should  be  the  cover-glass  (see  £  235). 

The  cover-glass  should  always  be  considerably  larger  than  the  object  0:0 
which  it  is  placed. 

§  222.  Cleaning  Cover-Glasses. — New  cover-glasses  should  be  put 
into  a  glass  dish  of  some  kind  containing  one  of  the  cleaning  mixtu 


142  SLIDES  AND  COVER-GLASSES.  [CH,   VI 7. 

(§  227)  and  allowed  to  remain  a  day  or  longer.  In  putting  them  in, 
push  one  in  at  a  time  and  be  sure  that  it  is  entirely  immersed,  otherwise 
they  adhere  very  closely,  and  the  cleaning  mixture  is  unable  to  act 
freely.  Soiled  covers  should  be  left  a  week  or  more  in  the  cleaning 
mixture.  An  indefinite  sojourn  in  the  cleaner  does  not  seem  to  injure 
the  slides  or  covers.  After  one  day  or  longer,  pour  off  the  cleaning 
mixture  into  another  glass  jar,  and  rinse  the  cover-glasses,  moving  them 
around  with  a  gentle  rotary  motion.  Continue  the  rinsing  until  all  the 
cleaning  mixture  is  removed.  One  may  rinse  them  occasionally,  and 
in  the  meantime  allow  a  very  gentle  stream  of  water  to  flow  on  them,  or 
they  may  be  allowed  to  stand  quietly  and  have  the  water  renewed  from 
time  to  time.  When  the  cleaning  mixture  is  removed  rinse  the  covers 
well  with  distilled  water,  and  then  cover  them  with  50^  to  75%  alcohol. 

§  223.  Wiping  the  Cover- Glasses.  —  When  ready  to  wipe  the 
cover-glasses,  remove  several  from  the  alcohol  and  put  them  on  a. soft, 
dry  cloth,  or  on  some  of  the  lens  paper  to  let  them  drain.  Grasp  a 
cover-glass  by  its  edges,  cover  the  thumb  and  index  of  the  other  hand 
with  a  soft,  clean  cloth  or  some  of  the  lens  paper.  Grasp  the  cover  be- 
tween the  thumb  and  index  and  rub  the  surfaces.  In  doing  this  it  is 
necessary  to  keep  the  thumb  and  index  well  opposed  on  directly  oppo- 
site faces  of  the  cover  so  that  no  strain  will  come  011  it,  otherwise  the 
cover  is  liable  to  be  broken. 

When  a  cover  is  well  wiped,  hold  it  up  and  look  through  it  toward 
some  dark  object.  The  cover  will  be  seen  partly  by  transmitted  and 
partly  by  reflected  light,  and  any  cloudiness  will  be  easily  seen.  If  the 
cover  does  not  look  clear,  breathe  on  the  faces  and  wipe  again.  If  it  is 
not  possible  to  get  a  cover  clear  in  this  way  it  should  be  put  again  into 
the  cleaning  mixture. 

As  the  covers  are  wiped,  put  them  in  a  clean  paper  box.  Handle 
them  always  by  their  edges,  or  use  fine  forceps.  Do  not  put  the  fingers 
on  the  faces  of  the  covers,  for  that  will  surely  cloud  them.  Wood-pulp 
paper,  from  which  most  of  the  boxes  are  now  made,  constantly  sheds 
particles  into  the  boxes  and  thus  soils  the  covers  stored  in  them.  This 
can  be  largely  obviated  by  coating  the  inside  of  the  boxes  with  a  thin 
solution  of  shellac. 

§  224.  Cleaning  Large  Cover-Glasses. — For  serial  sections  and 
especially  large  sections,  large  quadrangular  covers  are  used.  These 
are  to  be  put  one  by  one  into  cleaning  mixture  as  for  the  smaller  covers 
and  treated  in  every  way  the  same.  In  wiping  them  one  may  proceed 
as  for  the  small  covers,  but  special  care  is  necessary  to  avoid  breaking 
them.     A  safe  and  good  way  to  clean  the  large  covers  is  to  take  two 


CH.    17/.] 


SLIDES  AND  COVER  GLASSES. 


'  \l 


perfectly  flat,  smooth  blocks,  considerably  larger  than  the  cover-glasses. 
These  blocks  are  covered  with  soft  clean  cloth,  or  with  several  thick- 
nesses of  the  lens  paper  ;  if  now  the  cover-glass  is  placed  <>n  the-  one 
block  and  rubbed  with  the  other,  the  cover  may  be  cleaned  as  by  rub- 
bing its  faces  with  the  cloth-covered  finger  and  thumb.  It  is  especially 
desirable  that  these  large  covers  should  be  thin — not  over  l\,'\,  to 
mm. — otherwise  high  objectives  cannot  be  used  in  studying  the  prepa- 
rations. 

£  225.  Measuring  the  Thickness  of  Cover-Glasses. — It  is  of  the 
greatest  advantage  to  know  the  exact  thickness  of  the  cover-glass  on  an 
object  ;  for,  (a)  One  would  not  try  to  use  objectives  in  studying  the 
preparation  of  a  shorter  working  distance  than  the  thickness  of  the  cover 
(§57);  (b)  In  using  adjustable  objectives  with  the  collar  graduated 
for  different  thicknesses  of  cover,  the  collar  might  be  set  at  a  favorable 
point  without  loss  of  time  ;  (c)  For  unadjustable  objectives  the  thick- 
ness of  cover  may  be  selected  corresponding  to  that  for  which  the  object- 
ive was  corrected  (see  table,  $  27).  Furthermore,  if  there  is  a  varia- 
tion from  the  standard,  one  may  remedy  it,  in  part  at  least,  by  length- 
ening the  tube  if  the  cover  is  thinner,  and  shortening  it  if  the  cover  is 
thicker  than  the  standard  (§  96; . 

In  the  so-called  No.  1  cover-glasses  of  the  dealers  in  microscopical 
supplies,  the  writer  has  found  covers  varying  from  Tyo  mm.  to  vV,/  mm. 
To  use  cover-glasses  of  so  wide  a  variation  in  thickness  without  know- 
ing whether  one  has  a  thick  or  thin  one  is  simply  to  ignore  the  funda- 
mental principles  on  which  correct  microscopic  images  are  obtained. 


Fig.  120.   Micrometer  Calipers  (Brown  and  Sharpe).     Pocket  Calipers,  gradu- 
ated in  inches  or  millimeters,  and  well  adapted  for  measuring  coz/er-glas 

It  is  then  strongly  recommended  that  every  preparation  shall  be  cov- 
ered with  a  cover-glass  whose  thickness  is  known,  and  that  this  thick- 
ness should  be  indicated  in  some  way  on  the  preparation. 

>j  226.  Cover-Glass  Measurers.  —  For  the  purpose  of  measuring 
cover-glasses  there  are  three  very  excellent  pieces  of  apparatus.      The 


144 


SLIDES  AND  COVER-GLASSES. 


[CH.    VII. 


micrometer  calipers,  used  chiefly  in  the  mechanic  arts,  is  convenient,  and 
from  its  size  easily  carried  in  the  pocket.  The  two  cover-glass  meas- 
urer-, sp  scially  designed  for  the  purpose,  are  shown  in  Figs.  120-122. 
With  either  of  these  the  covers  may  be  more  rapidly  measured  than  with 

the  calipers. 

With  all  of  these  measurers  or  gauges  one  should  be  certain  that  the 
index  stands  at  zero  when  at  rest.  If  the  index  does  not  stand  at  zero 
it  should  be  adjusted  to  that  point,  otherwise  the  readings  will  not  be 
correct. 


Fig.  121.  Cover  Glass  Measurer  (Edward  Bausch). 

The  cover  glass  is  placed  in  the  notch  between  the  tivo  screws,  and  the  drum  is 
turned  by  the  milled  head  at  the  right  till  the  cover  is  in  contact  with  the  scrervs. 
The  thickness  is  then  indicated  by  the  knife  edge  on  the  drum  and  may  be  read  off 
directly  in  r^6th  mm.  or  x,is,jth  inch.  In  other  columns  is  given  the  proper  tube- 
length  for  various  unadjustable  objectives  ( j,  A,  \,  and  fa  in. )  made  by  the  Bausch 
and  Lor/ib  Optical  Company. 

As  the  covers  are  measured  the  different  thicknesses  should  be  put 
into  different  boxes  and  properly  labeled.  Unless  one  is  striving  for 
the  most  accurate  possible  results,  cover-glasses  not  varying  more  than 


CM.    J'//] 


SLIDES  AND  COVER  CLASSES. 


145 


,,'„  mm.  may  1>.-  put  in  the  same  box.      For  example,  it'  one  takes   ,',,■„ 

mm.  as  a  standard,  covers  varying  ,  r, ,,  mm.  on   each   side  may   lie  put 

into  the  same  h  >x.     In  thi>  case  tlie  box  would  contain  covers  of 

i 


II  ]  r,  1  6 

I     (HI   <        I    II   II   '        Mi    ri 


and  ,',,'„  i 


in'..  i22.  Zeiss  Cover-Gla 
urer.     II  'ill/  lliis  the  knife  edge  fa 
are  opened  by  means  of  a  lever,  and 
the  cover   inserted.      '1  he   thick) 
may  then  he  read  off  on  the  fact 
the  pointer  indicates  the  thickness  in 
hundredths  millimeter  in  the  outt  > 
circle  and  in  hundredths  inch  on  the 
:^_  inner  circle. 


S  227.  Cleaning  Mixtures  for  Glass. — The  cleaning  mixtures  used 
for  cleaning  slides  and  cover-glasses  are  those  commonly  used  in  chem- 
ical laboratories  : 

(  A)   Dichromate  of  Potash  and  Sulphuric  Acid. 

Bichromate  of  potash  (K,  Cr.2  07 )  200  grains 

Water,  distilled  or  ordinary  -  1000  cc. 

Sulphuric  acid  (H,  S04)  -  1000  cc. 

Dissolve  the  dichromate  in  the  water  by  the  aid  of  heat.  Pour  the 
solution  into  a  bottle  that  has  heeu  warmed  and  surrounded  by  a  wet 
towel.  Add  slowly  and  at  intervals  the  sulphuric  acid.  It  is  safer  to 
mix  the  ingredients  in  an  agate-ware  basin,  and  put  into  the  bottle  only 
after  the  mixture  is  cool. 

For  making  this  mixture,  ordinary  water,  commercial  dichromate  and 
strong  commercial  sulphuric  acid  should  be  used.  It  is  not  necessary 
to  employ  chemically  pure  materials. 

This  is  a  very  excellent  cleaning  mixture,  and  is  practically  odork-- 
It  is  exceedingly  corrosive  and  must  be  kept  in  glass  vessels.      It  may 
be  used  more  than   once,   hut  when  the  color  changes  markedly  from 
that  seen  in  the  fresh  mixture  it  should  be  thrown  away. 

{Bj   Sulphuric  and  Nitric  Acid  Mixture. 

Nitric  acid  (H  N03)  -  200  cc. 

Sulphuric  acid  (H.2S04)  -    300  cc. 

The  acids  should  be  strong,  but  they  need  not  be  chemically  pure. 

The  two  acids  are  mixed  slowly,  and  kept  in  a  glass-stoppered  bottle. 

This  is  a  more  corrosive  mixture  than   (A),  and  has  the  undesirable 

feature  of  giving  off  very  stilling  fumes,  therefore  it  must  be  carefully 

10 


146 


MOUNTING  AND  LABELING. 


[CM.    VII. 


covered.  It  may  be  used  several  times.  It  acts  more  rapidly  than  the 
dichromate  mixture,  but  on  account  of  the  fumes  is  not  so  well  adapted 
for  general  laboratories. 

MOUNTING,     AND    PERMANENT     PREPARATION    OF    MICROSCOPICAL 

OBJECTS. 

;i  228.  Mounting  a  Microscopical  Object  is  so  arranging  it  upon 
some  suitable  support  (glass  slide)  and  in  some  suitable  mounting  me- 
dium that  it  may  be  satisfactorily  studied  with  the  microscope. 

The  cover-glass  on  a  permanent  preparation  should  always  be  consider- 
ably larger  than  the  object;  and  where  several  objects  are  put  under  one 
cover-glass  it  is  false  economy  to  crotvd  them  too  closely  together. 

§  229.  Temporary  Mounting. — For  the  study  of  living  objects,  like 
amoebae,  white  blood  corpuscles,  and  many  other  objects  both  animal 
and  vegetable,  their  living  phenomena  can  best  be  studied  by  mounting 
them  in  the  natural  medium.  That  is,  for  amoebae,  in  the  water  in 
which  they  are  found  ;  for  the  white  blood  corpuscles,  a  drop  of  blood 
is  used  and,  as  the  blood  soon  coagulates,  they  are  in  the  serum.  Some- 
times it  is  not  easy  or  convenient  to  get  the  natural  medium,  then  some 
liquid  that  has  been  found  to  serve  in  place  of  the  natural  medium  is 
used.  For  many  things,  water  with  a  little  common  salt  (water  100  cc. , 
common  salt  yiyths  gram)  is  employed.  This  is  the  so-called  normal 
salt  or  saline  solution.  For  the  ciliated  cells  from  frogs  and  other  am- 
phibia, nothing  has  been  found  so  good  as  human  spittle.  Whatever 
is  used,  the  object  is  put  on  the  middle  of  the  slide  and  a  drop  of  the 
mounting  medium  added,  and  then  the  cover-glass.  The  cover  is  best 
put  on  with  fine  forceps,  as  shown  in  Fig.  123.  After 
the  cover  is  in  place,  if  the  preparation  is  to  be  studied 
for  some  time,  it  is  better  to  avoid  currents  and  evapora- 
tion by  painting  a  ring  of  castor  oil  around  the  cover  in 
such  a  way  that  part  of  the  ring  will  be  on  the  slide 
and  part  on  the  cover  (Fig.  140). 

Fio.  123.  To  shoiu  the  method  0/ putting  a  cover-glass  upon  a 
microscopic  preparation.  The  cover  is  grasped  by  one  edge,  the 
opposite  edge  is  then  brought  doivn  to  the  slide,  and  the  cover 
gradually  lozuered  upon  the  object. 


Fig.  124.  Needle  Holder  {Queen  &  Co.).  By  means  of  the 
screw  clamp  or  chuck  at  one  end,  the  needle  may  be  quickly 
changed. 


Fig.  123. 


CH.   Vll]  MOl7X/7N(;  AND  LABELING.  [47 

£  230.   Permanent    Mounting.—  For    making    permanent    mi< 
scopical    preparations,   there  are  three  great  methods.     Special  metn 
ods  of  procedure  arc  necessary  to  mount  objects  successfully  in  each  of 
these  ways.     The  best  mounting  medium  and  the  best  method  of  mount 
ing  in  a  given  case  ran  only  be  determined  by  experiment.     In  most 
cases  some  previous  observer  ha-,  already  made  the  necessary  experi 
ments  and  furnished  the  desired  information. 

The  three  methods  are  the  following  :  (A)  Dry  or  in  air  (§  231 )  ; 
(  B  1  ///  some  medium  miscible  with  waterJ  as  glycerin  or  glycerin  jelly 
I ?;  235  1  ;  (C)  In  some  resinous  medium  like  Jam  mar  or  Canada  balsam 
(§  240). 

§231.  Mounting  Dry  or  in  Air. — The  object  should  be  thoroughly 
dry.  If  any  moisture  remains  it  is  liable  to  cloud  the  cover-glass,  and 
the  specimen  may  deteriorate.  As  the  specimen  must  be  sealed,  it  is 
necessary  to  prepare  a  cell  slightly  deeper  than  the  object  is  thick. 
This  is  to  support  the  cover-glass,  and  also  to  prevent  the  running  in 
by  capillarity  of  the  sealing  mixture. 

ORDER    OF    PROCEDURE    IN    MOUNTING    OBJECTS    DRY    OR    IN"    AIR. 

i.  A  cell  of  some  kind  is  prepared.  It  should  be  slightly  deeper  than 
the  object  is  thick  (  $  233). 

2.  The  object  is  thoroughly  dried  (desiccated)  either  in  dry  air  or  by 
the  aid  of  gentle  heat. 

3.  If  practicable  the  object  is  mounted  on  the  cover-glass  ;  if  not  it  is 
placed  in  the  bottom  of  the  cell. 

4.  The  slide  is  warmed  till  the  cement  forming  the  cell  wall  is  some- 
what sticky,  or  a  very  thin  coat  of  fresh  cement  is  added  ;  the  cover  i- 
warmed  and  put  on  the  cell  and  pressed  down  all  around  till  a  shining 
ring  indicates  its  adherence  (§  234). 

5.  The  cover-glass  is  sealed  ($  234). 

6.  The  slide  is  labeled  ($  292). 

7.  The  preparation  is  cataloged  and  safely  stored  (§  293,  296). 

£  232.  Example  of  Mounting  Dry,  or  in  Air. — Prepare  a  shal 
low  cell  and  dry  it  (^  233).  Select  a  clean  cover-glass  slightly  larf 
than  the  cell.  Pour  upon  the  cover  a  drop  of  a  10',  solution  of  sali- 
cylic acid  in  95$  alcohol.  Let  it  dry  spontaneously.  Warm  the  slide 
till  the  cement  ring  or  cell  is  somewhat  sticky,  then  warm  the  covei 
gently  and  put  it  on  the  cell,  pressing  down  all  around  ^  231  ).  Seal 
the  cover,  label  and  catalog  1  ^  234,  292,  293  1. 

A  preparation  of  mammalian  red  blood  corpuscles  may  be  made  very 
satisfactorily  by  spreading  a  very  thin  layer  of  fresh  blood  on  a  covei 


1 48  MOL  rN TIN(  ■   A ND  LABEL ING.  [CH.   I  'II. 

with  the  end  of  a  slide.  After  it  is  dry,  warm  gently  to  remove  the  last 
tract.-  of  moisture  and  mount  precisely  as  for  the  crystals.  One  can  get 
the  blood  as  directed  for  the  Micro-spectroscopic  work  ( £  201). 


FlG.  125.  Turn -Table  for  scaling  cover-glasses  and  making  shallow  mounting 
cells.     ( Queen  &  Co. ) 

§  233.  Preparation  of  Mounting  Cells. — (  A  )  Thin  Cells.  These 
are  most  conveniently  made  of  some  of  the  microscopical  cements. 
Shellac  is  one  of  the  best  and  most  generally  applicable  (§316;.  To 
prepare  a  shellac  cell  place  the  slide  on  a  turn-table  (  Fig.  125)  and  cen- 
ter it,  that  is,  get  the  center  of  the  slide  over  the  center  of  the  turn-table. 
Select  a  guide  ring  on  the  turn-table  which  is  a  little  smaller  than  the 
cover-glass  to  be  used,  take  the  brush  from  the  shellac,  being  sure  that 
there  is  not  enough  cement  adhering  to  it  to  drop.  Whirl  the  turn-table 
and  hold  the  brush  lightly  on  the  slide  just  over  the  guide  ring  selected. 
An  even  ring  of  the  cement  should  result.  If  it  is  uneven,  the  cement 
is  too  thick  or  too  thin,  or  too  much  was  on  the  brush.  After  a  ring  is 
thus  prepared  remove  the  slide  and  allow  the  cement  to  dry  spontane- 
ously, or  heat  the  slide  in  some  way.  Before  the  slide  is  used  for 
mounting,  the  cement  should  be  so  dry  when  it  is  cold  that  it  does  not 
dent  when  the.  finger  nail  is  applied  to  it. 

A  cell  of  considerable  depth  may  be  made  with  the  shellac  by  adding 
successive  layers  as  the  previous  one  drys. 

(B)  Deep  Cells  are  sometimes  made  by  building  up  cement  cells,  but 
more  frequently,  paper,  wax,  glass,  hard  rubber,  or  some  metal  is  used 
for  the  main  part  of  the  cell.  Paper  rings,  block  tin  or  lead  rings  are 
easily  cut  out  with  gun  punches.  These  rings  are  fastened  to  the  slide 
by  using  some  cement  like  the  shellac. 

§  234.  Sealing  the  Cover-Glass  for  Dry  Objects  Mounted  in 
Cells. — When  an  object  is  mounted  in  a  cell,  the  slide  is  warmed  until 
the  cement  is  slightly  sticky,  or  a  very  thin  coat  of  fresh  cement  is  put 
on.  The  cover-glass  is  warmed  slightly  also,  both  to  make  it  stick  to 
the  cell  more  easily,  and  to  expel  any  remaining  moisture  from  the  ob- 
ject.    When  the  cover  is  put  on  it  is  pressed  down  all  around  over  the 


CH.   I VI.] 


MOUNTING  AND  LABELING. 


'  »■» 


cell  until  a  shining  ring  appears,  showing  that  there  is  an  intimate  con 
tact.  In  doing  this  use  the  convex  part  of  the-  fine  forceps  <>r  some 
other  blunt,  smooth  object  ;  it  is  also  necessary  to  avoid  pressing  on  the 
cover  except  immediately  over  the  wall  of  the  cell  for  fear  of  breaking 
the  cover.  When  the  cover  is  in  contact  with  the  wall  of  cement  all 
around,  the  slide  should  be  placed  on  the  turn-table  and  carefully  ar- 
ranged so  that  the  cover-glass  and  cell  wall  will  be  concentric  with  the 
guide  rings  of  the  turn-table.  Then  the  turn-table  is  whirled  and  a 
ring  of  fresh  cement  is  painted,  half  on  the  cover  and  half 'on  the  cell 
wall  (Fig.  140).  If  the  cover-glass  is  not  in  contact  with  the  cell  wall  at 
any  point  and  the  cell  is  shallow,  there  will  be  great  danger  of  the  fresh 
cement  running  into  the  cell  and  injuring  or  spoiling  the  preparation. 

When  the  cover-glass  is  properly  sealed,  the  preparation  is  put  in  a 
safe  place  for  the  drying  of  the  cement.  It  is  advisable  to  add  a  fresh 
coat  of  cement  occasionally. 

S  235.  Mounting  Objects  in  Media  Miscible  with  Water.— 
Many  objects  are  so  greatly  modified  by  drying  that  they  must  be 
mounted  in  some  medium  other  than  air.  In  some  cases  water  with 
something  in  solution  is  used.  Glycerin  of  various  strengths,  and 
glycerin  jelly  are  also  much  employed.  All  these  media  keep  the  ob- 
ject moist  and  therefore  in  a  condition  resembling  the  natural  one. 
The  object  is  usually  and  properly  treated  with  gradually  increasing 
strengths  of  glycerin  or  fixed  by  some  fixing  agent  before  being  per- 
manently mounted  in  strong  glycerin  or  either  of  the  other  media. 

In  all  of  these  different  methods,  unless  glycerin  of  increasing 
strengths  has  been  used  to  prepare  the  tissue,  the  fixing  agent  is 
washed  away  with  water  before  the  object  is  finally  and  permanently 
mounted  in  either  of  the  media. 

For  glycerin  jelly  no  cell  is  necessary    unless  the   object    has   a   con 
siderable  thickness. 


Fig.  126.  Centering  Card.  .1  card 
with  slops  for  the  slide-  and  circles  in 
tin-  position  occupied  by  the  center  ,7 the 
slide.  If  the  slide  is  put  upon  such  a 
card  it  is  very  easy  to  arrange  the 
Object  SO  that  it  :vill  be  approximately  in 
the  center  of  the  slide.  (From  the  Mi- 
croscope, Dec,  [886.) 


£  236.   Order  of  Procedure  in  Mounting  Objects  in  Glycerin. 
1.   A  cell  must  be  prepared  on  the  slide  if  the  object  1-  of  considerable 
thickness  (§  233,  234  >. 


i50  MOISTING  AND  LABELING.  [CH.    VII. 

2.  A  suitably  prepared  object  (§  235)  is  placed  on  the  center  of  a  clean 
slide,  and  if  no  cell  is  required  a  centering  card  is  employed  to  facilitate 
the  centering  (  Fig.  126.) 

j.  A  drop  of  pure  glycerin  is  put  upon  the  object,  or  if  a  cell  is  used, 
enough  to  till  the  cell. 

4.  In  putting  on  the  cover-glass  it  is  grasped  with  fine  forceps  and 
the  under  side  breathed  on  to  slightly  moisten  it  so  that  the  glycerin 
will  adhere,  then  one  edge  of  the  cover  is  put  on  the  cell  or  slide  and 
the  cover  gradually  lowered  upon  the  object  (Fig.  123).  The  cover 
is  then  gently  pressed  down.  If  a  cell  is  used,  a  fresh  coat  of  cement 
is  added  before  mounting  (§234.) 

Fig.  127.  Slide  arid  cover  glass  showing  method  of  anch- 
oring a  cover-glass  with  a  glycerin  prepaj'ation  ivhen  no 
cell  is  used.  A  cover-glass  so  anchored  is  not  liable  to 
move  when  the  cover  is  being  sealed  (I  238). 

Fig.  128.  Glass  slide  zvith  cover-glass,  a  drop  of  reagent 
and  a  bit  of  absorbent  paper  to  show  method  of  irriga- 
tion (I  247,  248). 


&,  **   *■  K^ 


5.  The  cover-glass  is  sealed  (§  234). 

6.  The  slide  is  labeled  (§  292). 

•/.  The  preparation  is  cataloged  and  safely  stored  (§  293,  296). 
§  237.   Order  of   Procedure  in    Mounting  Objects    in    Glycerin 
Jelly. 

1.  Unless  the  object  is  quite  thick  no  cell  is  necessary  with  glycerin 
jelly. 

2.  A  slide  is  gently  warmed  and  placed  on  the  centering  card  (Fig. 
126)  and  a  drop  of  warmed  glycerin  jelly  is  put  on  its  center.  The 
suitably  prepared  object  is  then  arranged  in  the  center  of  the  slide. 

3.  A  drop  of  the  warm  glycerin  jelly  is  then  put  on  the  object,  or  if 
a  cell  is  used  it  is  filled  with  the  medium. 

4.  The  cover-glass  is  grasped  with  fine  forceps,  the  lower  side 
breathed  on  and  then  gradually  lowered  upon  the  object  (Fig.  123), 
and  gently  pressed  down. 

5.  After  mounting,  the  preparation  is  left  flat  in  some  cool  place  till 
the  glycerin  jelly  sets,  then  the  superfluous  amount  is  scraped  and 
wiped  away  and  the  cover-glass  sealed  with  shellac  (§  234,  248). 

6.  The  slide  is  labeled  (§  292). 

7.  The  preparation  is  cataloged  and  safely  stored  (§  296). 

§  238.  Sealing  the  Cover-Glass  when  no  Cell  is  used. — -(A) 
For  glyccri>i   mounted  specimens.     The  superfluous  glycerin  is  wiped 


CM-   17/.] 


MOUNTING  AND  LABELING. 


IS' 


away  as  carefully  as  possible  with  a  moist  cloth,  then  four  minute  drops 
of  cement  arc  placed  at  the  edge  of  the  cover  1  Pig.  127  1,  and  allowed  to 
harden  for  half  an  hour  or  more.     These  will  anchor  the  cover-glas 
then  the  preparation  may  be  put  on  the  turn-table  and  a  ring  of  cement 

put  around  the  edge  while  whirling  the  turn-table. 


Fig.  129.  A — Simple  form  of  moist  chamber  made  with  a  plate  and  bowl.  B, 
bowl  serving  as  a  bell  jar ;   P,  plate  containing  the  water  and  over  which  the  bowl 

is  inverted;  S,  slides  on  which  are  mounted  preparations  which  are  to  be  kept 
moist.  These  slides  are  seen  endwise  and  rest  upon  a  bench  made  by  cementing 
short  pieees  of  large  glass  tubing  to  a  strip  of  glass  of  the  desired  length  and  width. 

R — Two  cover-glasses  (C)  made  eccentric,  so  that  they  may  be  more  easily  sepa- 
rated by  grasping  the  projecting  edge. 

C — Slide  (S)  re////  projecting  cover-glass  C).  The  projection  of  the  cover  en- 
ables one  to  grasp  and  raise  it  without  danger  of  moving  it  on  the  slide  and  thus 
folding  the  substance  under  the  cover.     (From  Proc.  Anier.  Micr.  Soc,  1 S91 ). 

(B)  For  objects  in  glycerin  jelly ',  FarranVs  solution  or  a  resinous  me- 
dium.  The  mounting  medium  is  first  allowed  to  harden,  then  the  su- 
perfluous medium  is  scraped  away  as  much  as  possible  with  a  knife,  and 
then  removed  with  a  cloth  moistened  with  water  for  the  glycerin  jelly 
and  Farrant's  solution  or  with  alcohol,  chloroform  or  turpentine,  etc.. 
if  a  resinous  medium  is  used.  Then  the  slide  is  put  on  a  turn-table  anil 
a  ring  of  the  shellac  cement  added.  (C)  Balsam  preparations  may  be 
sealed  with  shellac  as  soon  as  they  are  prepared,  but  it  is  better  to  allow 
them  to  dry  for  a  few"  days.  One  should  never  use  a  cement  for  seal- 
ing preparations  in  balsam  or  other  resinous  media  unless  the  solvent 
of  the  cement  is  not  a  solvent  of  the  balsam,  etc.  Otherwise  the  cement 
will  soften  the  balsam  and  finally  run  in  and  mix  with  it.  and  partly  or 
wholly  ruin  the  preparation.  Shellac  is  an  excellent  cement  for  sealing 
balsam  preparations,  as  it  never  runs  in,  and  it  serve-  to  avoid  any  in- 
jury to  the  preparation  when  cedar  oil,  etc. .are  used  for  homogeneous 
immersion  objectives. 


152  MOUNTING  AND  LABELING.  \_CH.   VII 

§  239.    Example  of  Mounting  in  Glycerin  Jelly. — For  this  select 

ime  stained  and  isolated  muscular  fibres  or  other  suitably  prepared 
objects.  S  under  isolation  §  244).  Arrange  them  on  the  middle  of 
a  slide,  using  the  centering  card,  and  mount  in  glycerin  jelly  as 
directed  in  £  223.  Air  bubbles  are  not  easily  removed  from  glycerin 
jelly  preparations,  so  care  should  be  taken  to  avoid  them. 

§  240.  Mounting  Objects  in  Resinous  Media. — While  the  media 
miscible  with  water  offer  many  advantages  for  mounting  animal  and 
vegetable  tissues  the  preparations  so  made  are  liable  to  deteriorate.  In 
many  cases,  also,  they  do  not  produce  sufficient  transparency  to  enable 
one  to  use  high  enough  powers  for  the  demonstration  of  minute  details. 

By  using  sufficient  care  almost  any  tissue  may  be  mounted  in  a  resin- 
ous medium  and  retain  all  its  details  of  structure. 

For  the  successful  mounting  of  an  object  in  a  resinous  medium  it 
must  in  some  way  be  deprived  of  all  water  and  all  liquids  not  miscible 
with  the  resinous  mounting  medium.  There  are  two  methods  of  bring- 
ing this  about:  (A)  By  drying  or  desiccation  (§  241),  and  (  B)  by 
successive  displacements  (§  243). 

s'  241.  Order  of  Procedure  in  Mounting  Objects  in  Resinous 
Media  by  Desiccation  : 

1.  The  object  suitable  for  the  purpose  (fly's  wings,  etc.  )  is  thorough- 
ly dried  in  dry  air  or  by  gentle  heat. 

2.  The  object  is  arranged  as  desired  in  the  center  of  a  clean  slide  on 
the  centering  card  (Fig.  126). 

3.  A  drop  of  the  mounting  medium  is  put  directly  upon  the  object  or 
spread  on  a  cover-glass. 

4.  The  cover-glass  is  put  on  the  specimen  with  fine  forceps  (Fig. 
123  j,  but  in  no  case  does  one  breathe  on  the  cover  as  when  media  mis- 
cible with  water  are  used. 

5.  The  cover-glass  is  pressed  down  gently. 

6.  The  slide  is  labeled  (§  292). 

7.  The  preparation  is  cataloged  and  safely  stored  (§  293,  296). 

8.  Although  it  is  not  absolutely  necessary,  it  is  better  to  seal  the 
cover  with  shellac  after  the  medium  has  hardened  round  the  edge  of 
the  cover  (§  238  C). 

£  242.  Example  of  Mounting  in  Balsam  by  Desiccation. — Find 
a  fresh  fly,  or  if  in  winter,  procure  a  dead  one  from  a  window  sill  or  a 
spider's  web.  Carefully  remove  the  fly's  wings,  being  especially  care- 
ful to  keep  them  the  dorsal  side  up.  With  a  camel's  hair  brush  remove 
any  dirt  that  may  be  clinging  to  them.  Place  a  clean  slide  on  the  cen- 
tering card,  then  with  fine  forceps  put  the  two  wings  within  one  of  the 


CH.   III.]  MOUNTING  AND  LABELING  153 

guide  rings.  Leave  one  dorsal  side  up,  turn  the  other  ventral  side  up. 
Spread  some  Canada  balsam  on  the  face  of  the  cover  glass  and  with  the 
fine  forceps  place  the  cover  upon  the  wings  (Fig.  [23).  Probabl}  some 
air  bubbles  will  appear  in  the  preparation,  but  it"  the  slide  is  put  in  a 
warm  place  these  will  soon  disappear.     Label,  catalog,  etc.,  (§  -">i 

-•05). 

sj  243.  Mounting  in  Resinous  Media  by  a  Series  of  Displace- 
ments.— For  examples  of  this  see  the  procedure  in  the  paraffin  and  in 
the  collodion  methods  (£  265,  284).  The  first  step  in  the  -  :ries  is  D 
hydration,  that  is,  the  water  is  displaced  by  some  liquid  which  is  misei- 
ble  both  with  the  water  and  the  next  liquid  to  be  used.  Strong  alcohol 
(95'/f  or  stronger)  is  usually  employed  for  this.  Plenty  of  it  must  be 
used  to  displace  the  last  trace  of  water.  The  tissue  may  be  soaked  in  a 
dish  of  the  alcohol,  or  alcohol  from  a  pipette  may  be  poured  upon  it. 
Dehydration  usually  occurs  in  the  thin  objects  to  be  mounted  in  balsam 
in  5  to  15  minutes.  If  a  dish  of  alcohol  is  used  it  must  not  be  used  too 
many  times,  as  it  loses  in  strength. 

The  second  step  is  clearing.  That  is,  some  liquid  which  is  miscible 
with  the  alcohol  and  also  with  the  resinous  medium  is  used.  This 
liquid  is  highly  refractive  in  most  cases,  and  consequently  this  step  is 
called  clearing  and  the  liquid  a  clearer.  The  clearer  displaces  the  alco- 
hol, and  renders  the  object  more  or  less  translucent.  In  case  the  water 
was  not  all  removed,  a  cloudiness  will  appear  in  parts  or  over  the  whole 
of  the  preparation.  In  this  case  the  preparation  must  be  returned  to 
alcohol  to  complete  the  dehydration. 

One  can  tell  when  a  specimen  is  properly  cleared  by  holding  it  over 
some  dark  object.  If  it  is  cleared  it  can  be  seen  only  with  difficulty,  as 
but  little  light  is  reflected  from  it.  If  it  is  held  toward  the  window, 
however,  it  will  appear  translucent. 

77/e  third  and  final  step  is  the  displacement  of  the  clearer  by  the  resin 
ous  mounting  medium. 

The  specimen  is  drained  of  clearer  and  allowed  to  stand  for  a  short 
time  till  there  appears  the  first  sign  of  dullness  from  evaporation  of  the 
clearer  from  the  surface.  Then  a  drop  of  the  resinous  medium  is  put 
on  the- object,  and  finally  a  cover-.^iass  is  placed  over  it,  or  a  drop  of 
the  mounting  medium  is  spread  on  the  cover  ami  it  is  then  put  on  the 
object. 


154  ISOLATION  OF  ELEMENTS.  [CH.    VII. 

ISOLATION    OF    HISTOLOGICAL    ELEMENTS. 

ij  244.  For  a  correct  conception  of  the  forms  of  the  cells  and  fibers  of 
the  various  organs  of  the  body,  one  must  see  these  elements  isolated 
and  thus  be  able  to  inspect  them  from  all  sides.  It  frequently  occurs 
also  that  the  isolation  is  not  quite  complete,  and  one  can  see  in  the 
clearest  manner  the  relations  of  the  cells  or  fibers  to  one  another. 

The  chemical  agents  or  solutions  for  isolating  are,  in  general,  the 
same  as  those  used  for  hardening  and  fixing.  But  the  solutions  are 
only  about  one-tenth  as  strong  as  for  fixing,  and  the  action  is  very  much 
shorter,  that  is,  from  one  or  two  hours  to  as  many  days.  In  the  weak 
solution  the  cell  cement  or  connective  tissue  is  softened  so  that  the  cells 
and  fibers  may  be  separated  from  one  another,  and  at  the  same  time  the 
cells  are  preserved.  In  fixing  and  hardening,  on  the  other  hand,  the 
cell  cement,  like  the  other  parts  of  the  tissue,  are  made  firmer.  It  is 
better  also  to  dilute  the  fixing  agents  with  normal  salt  solution  (§  313  ) 
than  merely  with  water. 

§  245.  Isolation  by  Means  of  Formaldehyde. — Formaldehyde  in 
a  ,'-'„ '/(  solution  in  normal  salt  solution  is  one  of  the  very  best  dissociat- 
ing agents  for  brain  tissue  and  all  the  forms  of  epithelium  (  §  308).  It 
is  prepared  as  follows  :  5  cc.  of  formal,  formol,  formalin  or  formalose, 
that  is,  a  40'/  solution  of  formaldehyde,  are  mixed  with  995  cc.  of 
normal  salt  solution.  This  acts  quickly  and  preserves  delicate  struct- 
ures like  the  cilia  of  ordinary  epithelia,  and  also  of  the  endymal  cells  of 
the  brain.  It  is  very  satisfactory  for  isolating  the  nerve  cells  of  the 
brain.  For  the  epithelium  of  the  trachea,  intestines,  etc.,  the  action  is 
sufficient  in  two  hours  ;  good  preparations  may  also  be  obtained  after 
two  days  or  more.  The  action  on  nerve  tissue  of  the  brain  is  about  as 
rapid.  For  the  stratified  epithelia,  like  those  of  the  skin,  mouth,  etc., 
it  may  require  two  or  three  days  for  the  most  satisfactory  preparations. 
See  Figs.  130  and  131. 

^  246.  Example  of  Isolation. — Place  a  piece  of  the  trachea  of  a 
very  recently  killed  animal,  or  the  roof  of  a  frog's  mouth,  in  the  form- 
aldehyde dissociator.  After  two  hours  or  more,  up  to  two  or  three 
days,  excellent  preparations  of  ciliated  cells  may  be  obtained  by  scrap- 
ing the  trachea  or  roof  of  the  mouth  and  mounting  the  scrapings  on  a 
slide.  If  one  proceeds  after  two  hours,  probably  most  of  the  cells  will 
cling  together,  and  in  the  various  clumps  will  appear  cells  on  end  show- 
ing the  cilia  or  the  bases  of  the  cells,  and  other  clumps  will  show  the 
cells  in  profile.  By  tapping  the  cover  gently  with  a  needle  holder  or 
other  light  object  the  cells  will  be  more  separated  from  one  another,  and 
many  fully  isolated  cells  will  be  seen. 


CI  I.   VII] 


ISOLATIOX  OF  ELEMENTS. 


155 


S  247.  Staining  the  Cells. — Almost  any  stain  may  be  used  for  the 
formalin  dissociated  cells.  As  an  example,  one  may  use  1  osin 
This  may  be  drawn  under  the  cover  of  the  already  mounted  preparation 
(Fig.  12S),  or  a  new  preparation  may  be  made  and  the  scrapings 
mixed  with  a  drop  of  the  eosin  before  putting  on  the  cover-glass.  It  is 
an  advantage  to  study  unstained  preparations,  otherwise  one  may  obtain 
the  erroneous  opinion  that  the  structure  cannot  be  seen  unless  it  is 
stained.  The  stain  makes  the  structural  features  somewhat  plainer  ;  it 
also  accentuates  some  features  and  docs  not  affect  so  markedly  others 

$  248.    Permanent  Preparations  of  Isolated  Cells. — If  one  de- 
sires to  make  a  permanent  preparation   of  the  isolated  cells  it  may  be 
done  by  placing  a  drop  of  glycerin  at  the  edge  of  the  cover  and  allowing 
it  to  diffuse  under  the  cover,   or  the  diffusion  may  be  hurried  by  using 


Fig.  130.  Adjustable  lens  holder  with  universal  joint.     This  is  especially  useful 
for  gross  dissections,  and  for  dissecting  the  partly  isolated  elements  with  needles. 

a  piece  of  blotting  paper,  as  shown  in  Fig.  128.  One  may  also  make  a 
new  preparation  and  either  with  or  without  staining,  mix  the  cells  with 
a  drop  of  glycerin  on  the  slide  and  then  cover,  or  one  may  use  glycerin 
jelly  I  ^  239,  309 

£  249.    Isolation   of  Muscular  Fibers. — For  this  the  formal  dis 
ciator  may  be  used  (S  245,  308  I,  but  the  nitric  acid  method  is  more  sue 


IS6 


ISOLATION  OF  ELEMENTS. 


\CH.    VII. 


cessful  §312)  The  fresh  muscle  is  placed  in  this  in  a  glass  vessel. 
At  the  ordinary  temperature  of  a  sitting  room  (20  degrees  centigrade) 
the  connective  tissue  will  be  so  far  gelatinized  in  from  one  to  three  days 
that  it  is  very  easy  to  separate  the  fascicles  and  fibers  either  with  nee- 
dles <>r  by  shaking  in  a  test  tube  or  reagent  vial  (Fig.  132)  with  water. 
It  takes  longer  for  some  muscles  to  dissociate  than  others,  even  in  the 
same  temperature,  so  one  must  try  occasionally  to  see  if  the  action  is 
sufficient.      When  it  is,  the  acid  is  poured  off  and  the  muscle  washed 


Fig.  131.  Adjustable  lens  holder  for  the  same  purposes  as  Fig.  130. 
&  Lomb  Optical  Company). 


The  Bausch 


gently  with  water  to  remove  the  acid.  If  one  is  ready  to  make  the  prep- 
arations at  once  they  may  be  isolated  and  mounted  in  water.  If  it  is  de- 
sired to  keep  the  specimen  indefinitely,  or  several  days,  the  water  should 
be  poured  off  and  a  half  saturated  solution  of  alum  added  (§  299).  The 
alum  solution  is  also  very  advantageous  if  the  specimens  are  to  be 
stained.  The  specimens  may  be  mounted  in  glycerin,  glycerin  jelly  or 
balsam.     Glycerin  jelly  is  the  most  satisfactory,  however. 


CH    17/.]  COLLODION  SECTIONING.  157 

THE    PREPARATION   OF   SECTIONS   OF    TISSUES    AND   ORGANS. 

£  250.   At  the  present  time  there  are  three  principal  methods  of  ob 
taining  thin  sections  of  tissues  and  organs  for  microsc  >pi<  study.     'I  I 
methodsare:    The  Collodion  Method^  the  Paraffin  Method^  and  tin  I  ■ 
ing  Method.     Each  of  these  in  ithods  has  its  special  application,  although 
the  collodion  method  is  p  xhaps  the  most  generally  applicable,  and  the 
freezing   method  the  most  restricted,  and  is  used  mostly  in  pathological 
work,  where  rapid  diagnosis  is  necassary  and  the  finest  detail- of  struct 
tire  are  not  so  important.     With  the  paraffin  method  the  thinnesl 
tions  may  be  made,  and  in  some  ways  it  is  the  most  satisfactory  of  all. 
A  good  microtome  is  of  very  great  aid  in  sectioning. 

§251.  The  Collodion  Method.  — In  sectioning  by  this  method  the 
tissues  are  first  hardened  properly  and  then  entirely  infiltrated  with  col- 
lodion, and  the  collodion  hardened.  It  is  not  removed  from  the  tissue, 
hut  on  account  of  its  transparency  does  no  harm. 

^  252.  Fixing  and  Hardening  the  Tissue. — Any  of  the  approved 
methods  of  hardening  and  fixing  may  be  employed.  A  good  general 
method  which  is  applicable  to  nearly  all  of  the  tissues  and  organs  is  that 
by  Picric- Alcohol.  For  the  preparation  of  the  solution  sec  1  >;  315  .  A 
small  piece  of  tissue  or  organ  not  containing  more  than  two  to  three- 
cubic  centimeters  is  placed  in  40  or  50  cc.  of  the  picric-alcohol  and  left 
6  to  24  hours,  when  the  first  picric-alcohol  should  be  thrown  away  and 
fresh  added.  After  one  or  two  days  more  the  picric-alcohol  should  be 
poured  off  and  67'/  alcohol  added.  In  a  day  or  two  this  is  replaced  by 
75%  or  §2'/c  alcohol  ;  82'/  is  on  the  whole  most  satisfactory,  and  the 
tissue  may  be  left  in  this  till  it  is  read}-  for  dehydration. 

£  253.  Dehydration  before  Infiltration. — When  one  is  ready  to 
imbed  for  sections,  the  tissue  must  first  be  dehydrated  in  plentiful  Q« 
or  stronger  alcohol.  It  is  better  to  take  only  a  small  piece  for  this. 
The  smaller  the  piece  the  thinner  the  sections  may  lie  made.  The  de- 
hydration will  usually  be  completed  in  2  to  24  hours.  If  the  alcohol  is 
changed  two  or  three  times  the  dehydration  will  be  hastened. 

£  254.  Saturating  with  Ether- Alcohol  1  £  306). — The  next  step  is 
to  remove  the  tissue  from  the  alcohol  and  place  it  in  a  vial  of  ether- 
alcohol  (S  306)  for  2  to  24  hours.  The  dehydration  is  somewhat  m 
complete  by  this  step,  and  the  tissue  is  more  perfectly  prepared  for  the 
reception  of  the  collodion.  If  the  dehydration  is  very  thorough  in  the 
alcohol,  this  step  may  be  omitted,  however,  but  one  is  surer  of  sue 
if  the  ether-alcohol  is  used. 

£255.    Infiltration  with  Thin  Collodion.— The   ether  -  alcohol    i^ 
poured  off,  and  a  mixture  of  thin  collodion  is  added     s  304         Two  or 


158  COLLODION  SECTIONING.  [CH.    VII. 

three  hours  will  suffice  for  objects  two  or  three  millimeters  in  thickness. 
A  st. iv  of  one  or  more  days  does  no  harm.  The  larger  the  object  the 
more  time  is  needed. 

.^  256.  Infiltration  with  Thick  Collodion. — The  thin  collodion  is 
poured  off  and  thick  collodion  (§  304)  added.  For  very  small  objects, 
four  or  five  hours  will  suffice  to  infiltrate,  but  for  larger  objects  a  longer 
time  is  necessary.  The  tissue  does  not  seem  to  be  injured  at  all  in  the 
thick  collodion,  and  a  stay  in  it  during  a  day  or  even  a  week  is  more 
certain  to  insure  a  perfect  infiltration. 

;i  257.  Imbedding. — The  tissue  may  be  imbedded  in  a  paper  box, 
such  as  is  used  for  paraffin  imbedding,  or  in  any  of  the  other  boxes  de- 
vised for  paraffin.  It  is  better,  if  paper  is  used,  to  put  a  very  small 
amount  of  oil  on  the  paper  to  prevent  the  collodion  from  sticking  to  it. 
Vaselin  spread  over  lightly  and  then  all  removed,  so  far  as  possible, 
with  a  cloth  or  with  lens  paper,  gives  the  right  surface.  For  small  ob- 
jects it  is  more  convenient  to  imbed  immediately  on  a  holder  that  may 
be  clamped  into  the  microtome.  Cylinders  or  blocks  of  glass,  vulcanite, 
wood  and  cork  have  all  been  recommended  and  used.  A  cork  of  the 
proper  size  is  most  convenient,  and  for  many  purposes  answers  well. 
Some  collodion  is  put  on  the  end  of  the  cork  and  a  pin  put  near  one  edge. 
The  tissue  is  transferred  from  the  thick  collodion  to  the  cork  and  leaned 
against  the  pin.  Drops  of  the  thick  collodion  are  then  poured  on  the 
tissue,  and  by  moving  the  cork  properly  the  thick,  viscid  mass  may  be 
made  to  surround  and  envelop  the  tissue.  Drops  of  collodion  are  added 
at  short  intervals  until  the  tissue  is  well  surrounded,  and  then  as  soon 
as  a  slight  film  hardens  on  the  surface,  the  cork  bearing  the  tissue  is 
inverted  in  a  wide-mouth  vial  of  considerably  larger  diameter  than  the 
cork  (Fig.  132).  The  vial  should  contain  sufficient  chloroform  to  float 
the  cork.  The  vial  is  then  tightly  corked.  In  imbedding  somewhat 
larger  objects  on  the  end  of  a  cork  or  other  holder,  it  is  frequently  ad- 
vantageous to  wind  oiled  paper  around  the  holder  or  cork,  tie  it  tightly 
and  have  the  projecting  hollow  cylinder  sufficiently  long  to  receive  the 
object.  The  tissue  is  then  put  into  the  cylinder  and  sufficient  collodion 
added  to  completely  immerse  it.  As  soon  as  a  film  has  formed  over  the 
exposed  end,  the  cork  may  be  inverted  and  immersed  in  chloroform,  as 
described  above. 

§  258.  Hardening  and  Clarifying  the  Collodion. — After  a  few 
hours  the  collodion  is  hardened  by  the  chloroform.  If  it  acts  long 
enough  the  imbedding  mass  is  rendered  entirely  transparent,  if  110  water 
is  present.      Whenever  the  collodion  is  hard,  whether  it  is  clear  or  not, 


<//.    17/.] 


COU.OMOS  SECTIONING. 


the  chloroform  is  poured  off  and  the  carbol  xylene*  clarifies  (§  30 
added.  In  a  few  hours  the  imbedded  mass  will  become  as  transparent 
glass  and  the  tissue  will  seem  to  have  nothing  around  it.  Sometimes 
the  collodion  remains  white  and  opaque  for  a  considerable  time.  So  far 
as  the  writer  has  been  able  to  judge,  this  is  due  to  moisture,  [f  one 
breathes  on  the  mass  too  much  while  imbedding,  <>r  if  it  is  very  damp 
in  the  room,  the  opacity  may  result.  Sometimes,  in  objects  of  consid- 
erable size,  this  may  remain  for  a  week.  This  is  the  exception,  how- 
ever, and  if  the  mass  seems  sufficiently  hard  and  tough,  the  cutting  may 
proc.ed  even  if  the  clarification  is  incomplete.  ! 


■~--~  -  ■ 


Fig.  132.  Preparation   Vials  for  Histology  and  Embryology.     These  represent 

the  two  vials,  natural  size,  that  have  been  found  most  useful.      They  are  kept  in 
blocks  rc'iih  holes  0/  the  proper  size. 

In  case  the  imbedding  mass  will  not  clarify  after  a  few  days  the  im- 
bedded object  may  be  placed  in  95'/,  alcohol  for  a  day  For  dehydration, 
and  then  passed  through  chloroform  and  into  the  clarifier.  There  is 
usually  no  trouble  in  getting  the  mass  perfectly  clear  in  this  way. 

*The  hydrocarbon  xylene  (Cs  HI())  is  called  xylol  in  German.  In  English,  mem- 
bers of  the  hydrocarbon  series  have  the  termination  "  ene,"  while  members  of  the 
alcohol  series  terminate  in  "ol." 

fThe  imbedded  object  may  remain  in   the  castor  xylene  clarifier  indefin 
without  harm.     The  collodion  grows  somewhat  toucher  by  a  prolonged  stay  in  it. 
After  cutting  all  the  sections  desired  at  one  time,  the  imbedded  tissue  is  returned 
to  the  clarifier  for  future  sections. 


160  COLLODION  SECTIONING.  [CH.    VI L 

$  259.   Cutting  the  Sections. — For  collodion  sectioning  a  long,  draw- 
ing cut  is  necessary  in  order  to  obtain  thin,   perfect  sections.     The  ob- 

t  is,  therefore,  put  in  the  jaws  of  the  microtome  at  the  right  level, 
and  the  knife  arranged  so  that  half  or  more  of  the  blade  of  the  knife  is 
used  in  cutting  the  section.  It  is  advantageous  also  to  have  the  object 
placed  with  its  long  diameter  parallel  with  the  edge  of  the  knife.  The 
surrounding  collodion  mass  should  be  cut  away,  as  in  sharpening  a  lead 
pencil,  so  that  there  is  not  more  than  a  thickness  of  about  two  millime- 
ters all  aionnd  the  tissue.  This  is  to  render  the  diameter  of  the  ena  to 
be  cut  as  small  as  possible.  The  smaller  the  object  the  thinner  can  the 
sections  be  made.  With  an  object  two  to  three  millimeters  thick  and 
not  over  five  millimeters  wide,  and  a  good  sharp  knife,  sections  5 /a  to 
6/j.  can  be  cut  without  difficulty.  When  knife  and  tissue  are  properly 
arranged  the  tissue  is  well  wet  and  the  knife  flooded  with  the  clarifier. 
Make  the  sections  with  a  steady  motion  of  the  knife.  Then  draw  the 
section  up  toward  the  back  of  the  knife  with  an  artist's  brush  and  make 
the  next  section.  Arrange  the  sections  in  serial  order  on  the  knife- 
blade  till  enough  are  cut  to  fill  the  area  that  the  cover-glass  will  cover. 

§  260.  Transferring  the  Sections  to  the  Slide. — If  the  clarifier 
has  evaporated  so  as  to  leave  the  sections  somewhat  dry  on  the  knife, 
add  a  small  amount.  Take  a  piece  of  thin  absorbent,  close-meshed  pa- 
per* about  twice  the  size  of  a  slide  and  place  it  directly  upon  the  sec- 
tions. Press  the  paper  down  evenly  all  around  and  then  pull  the  paper 
off  the  edge  of  the  knife. t  The  sections  will  adhere  to  the  paper.  Place 
the  paper,  sections  down,  on  a  slide,  taking  care  that  the  sections  are  in 
the  desired  position  on  the  slide.  Use  some  ordinary  lens- paper  or  any 
absorbent  paper,  and  press  it  down  gently  upon  the  transfer  paper. 
This  will  absorb  the  oil,  and  then  the  transfer  paper  may  be  lifted,  with 
a  rolling  motion,  from  the  slide.     The  sections  will  remain  on  the  slide. 

.^  261.    Fastening  the   Sections  to  the   Slide. — Drop  just  enough 


*  Various  forms  of  paper  have  been  used  to  handle  the  collodion  sections.  It 
should  be  moderately  strong,  fine  meshed  and  not  liable  to  shed  lint,  and  fairly  ab- 
sorbent. One  of  the  first  and  most  successful  papers  recommended  is  "closet  or 
toilet  paper."  Cigarette  paper  is  also  excellent.  In  my  own  work  the  silky  Japa- 
nese paper,  called  "  Usago  "  paper,  has  been  found  almost  perfect  for  the  purpose. 
Ordinary  lens  paper  or  thin  blotting  paper  for  absorbing  the  oil  is  used  with  it. 

t  If  one  is  a  long  time  cutting  a  series  of  sections,  it  sometimes  occurs  that  the 
xylene  evaporates,  and  while  the  sections  may  not  look  dry,  they  are  practically 
in  castor  oil  and  not  easily  transferable.  In  such  a  case  fresh  clarifier  or  even 
a  little  xylene  to  thin  the  oil  on  the  sections  may  be  used.  If  the  oil  is  too  thick 
it  is  viscid  and  there  is  difficulty  in  handling  the  sections  with  the  paper  as  they 
stick  rather  firmly  to  the  knife. 


CH.   171}  COLLODION  SECTIONING. 

ether-alcohol  (equal  parts  of  sulphuric  ether  and  95$  alcohol  1  on  the 
sections  to  moisten  them.  This  will  melt  the  collodion  and  fasten  tin- 
sections  to  the  slide.  Allow  the  slide  to  remain  in  the  air  till  the  sur- 
face begins  to  look  slightly  dull  or  glazed. 

Sometimes,  especially  when  the  air  is  moist,  the  sections  wrinkle 
badly  when  the  ether-alcohol  is  put  on  to  fasten  them  to  the  slide. 
The  excessive  wrinkling  can  be  avoided  by  using  one  part  alcohol  and 
two  parts  ether  instead  of  using  equal  parts  of  each.  Perhaps  also  it 
would  be  advantageous  in  this  case  to  use  absolute  alcohol. 

Fig.  133.  Pipette  for  adding  liquids  drop- 
wise  and  for  washing  preparations.  (  Whit- 
all,   Tat  it  m  CV  Co.) 

§  262.  Removing  the  Oil  from  the  Sections.  —  As  soon  as  the 
ether-alcohol  has  evaporated  sufficiently  to  leave  the  surface  dull,  place 
the  slide  in  a  jar  of  ordinary  commercial  benzin.  It  may  be  left  here  a 
day  or  more  without  injury  to  the  sections,  but  if  moved  around  in  the 
jar  the  oil  will  be  removed  in  three  to  five  minutes.  From  the  benzin 
transfer  to  a  jar  of  95^  alcohol  to  wash  away  the  benzin.  One  may 
use  alcohol  in  the  beginning,  but  it  dissolves  the  oil  far  less  rapidly  than 
the  benzin.  The  slide  may  remain  in  the  alcohol  half  a  day  or  more  if 
one  wishes,  but  a  stay  of  five  minutes  or  a  thorough  rinsing  of  half  a 
minute  or  so  by  moving  the  slide  around  in  the  alcohol  will  suffice. 

£  263.  Staining  the  Sections  with  an  Alcoholic  Stain.  —  If  an 
alcoholic  stain  containing  50$  or  more  alcohol  (for  example,  hydro- 
chloric acid  carmine  in  70^  alcohol)  is  used,  the  slide  may  be  removed 
from  the  gsf/r  alcohol,  drained  somewhat  and  then  the  stain  poured 
upon  the  sections,  or  preferably,  the  slide  immersed  in  a  jar  of  the  stain. 
The  stain  is  finally  washed  away  with  67'/  or  stronger  alcohol,  the  sec- 
tions dehydrated  in  95$  alcohol,  cleared  and  mounted  in  balsam. 

£  264.  Staining  the  Sections  with  an  Aqueous  Dye. — In  staining 
with  a  watery  stain,  the  slide  bearing  the  sections  is  transferred  from 
the  95'/  alcohol  and  plunged  into  a  jar  of  water,  and  either  allowed  to 
remain  a  few  minutes  or  moved  around  in  the  water  a  moment.  Then 
it  is  placed  horizontally,  and  some  of  the  stain  placed  on  the  secti< 
with  a  pipette,  or  preferably,  it  is  immersed  in  a  jar  of  the  stain  ;  in 
case  of  immersion,  however,  the  slide  should  stand  vertically  or  nearly 
so,  then  any  particles  of  dust,  etc.,  in  the  stain  will  settle  to  the  bottom 
of  the  vessel  and  not  settle  on  the  sections.  When  the  sections  are 
stained,  usually  within  five  minutes,  they  are  thoroughly  washed  with 
water  either  by  the  use  of  a  pipette  or  preferably  by  immersing  in  a  jar 
1 1 


l62 


COLLODION  SECTIONING. 


[CU.    VII. 


of  water.  They  may  then  be  counterstaiued  for  half  a  minute  with 
some  general  dye,  like  eosin  or  picric  acid,  or  mounted  with  but  the  one 
stain.* 


Fig.  135. 


Fig.  1^6. 


Fig  134.  Waste  Boivl  with  rack  for  supporting  slides  and  a  small  funnel  in 
which  the  slides  stand  while  draining.  This  outfit  is  easily  made  by  any  tin  smith. 
The  rack  is  composed  of  two  brass  rods  about  j  mm.  in  diameter.  The  bent  end 
pieces  are  sheet  brass.  The  funnel  is  made  of  tin,  copper  or  brass.  Either  copper 
or  brass  is  preferable  to  tin.  A  glass  dish  like  that  shoivn  in  Fig.  135  is  better  than 
a  bowl,  as  it  can  be  more  readily  and  thoroughly  cleaned.  {Cut  loaned  by  Wm. 
Wood  &  Co.). 

Fig.  135.  Round  glass  aquarium.  This  glass  vessel  is  better  than  the  bowl  for 
all  the  uses  described  for  the  boivl.     (  W/niall,  Tatum  &  Co. ) 

Fig.  136.  Glass  box  with  cover.  These  boxes  may  be  had  of  various  sizes  and 
can  be  used  advantageously  for  water,  and  for  cleaning  mixture  for  slides  and 
cover glasses  {\  227).     {Whiiall,  Tatum  &  Co.) 


*  In  the  past  the  plan  for  changing  sections  from  95%  alcohol  to  water,  for  ex- 
ample, has  been  to  run  them  down  gradually,  using  75,  50  and  35%  alcohol  suc- 
cessively. Each  percentage  may  vary,  but  the  principle  of  a  gradual  passing  from 
strong  alcohol  to  water  was  advocated.  On  the  other  hand,  I  have  found  that  the 
safest  method  is  to  plunge  the  slide  directly  into  water  from  the  95%  alcohol.  The 
diffusion  currents  are  almost  or  quite  avoided  in  this  way.  There  is  no  time  for 
the  alcohol  and  water  to  mix,  the  alcohol  is  washed  away  almost  instantly  by  the 
flood  of  water.  So  in  dehydrating  after  the  use  of  watery  stains,  the  slide  is 
plunged  quickly  into  a  jar  of  95%  alcohol.  The  diffusion  currents  are  avoided  in 
the  same  way,  for  the  water  is  removed  by  the  flood  of  alcohol.  This  plan  lias 
been  submitted  to  the  severe  test  of  laboratory  work,  and  has  proved  itself  perfectly 
satisfactorv. 


C/7.    17/.] 


cou.omox  s/:<  'TIONING. 


ORDER  OF  PROCEDURE    IN'    MAKING    MICROSCOPICAL    PREPARATIONS   HY 

Till'.    COLLODION    MKTIIOI). 

i;  265.    It  will  he   seen    from  this   table,   and    suctions    252  'hat 

it  requires  about  five  days  to  get  a  microscopical  preparation  if  one  com 
mences  with  the  fresh  tissue.     Other  methods  of  hardening  might  re- 
quire as  many  months.      It  is  evident,  therefore,  that  one  must  exercise 
foresight  in  histology  or  much  time  will  be  wasted. 


1.  Fixing   and    hardening    the    tissues 

(g  252),  4  days  or  more. 

2.  Dehydrating  the  ohject  to  he  cut  in 

95%  or  stronger  alcohol  (?  253),   2- 
24  hours. 

3.  Saturating  the  tissue  in  ether-alcohol 

(§  254),  2-24  hours. 

4.  Infiltrating  with    thin    collodion 

{\  255),  2  hours  to  2  days. 

5.  Infiltrating  in  thick  collodion  (I  256), 

5  hours  to  several  days. 

6.  Iinhedding  the  tissue  (#  257),     15  to 

20  minutes. 

7.  Hardening  the  collodion  with  chlo- 

roform (  g  258),  5-24  hours.     . 

8.  Clarifying  and  further  hardening  the 

collodion  with  castor-xylene  (§  25S), 
10-36  hours. 

9.  Cutting  the  sections  {\  259),     10  min- 

utes to  2  hours. 

10.  Transferring  the  sections  to  a  slide 

with  paper  {},  260),  1  minute. 

11.  Fastening  the  sections  to  the  slide 

with  ether- alcohol  (\  261),     1  or  2 
minutes. 

12.  Removing  the  oil   from  the  sections 

with  benziu  and  alcohol  ($  262),  3-5 
minutes,  or  24  hours. 


13.  Staining  the  sections  witli  an   alco- 

holic dye  ('i  263-264),    2  minutes  to 
24  hours. 

14.  Staining  the  sections  with  an  aque- 

ous dye  (?  264),  2-10  minutes. 

15.  Removing    the   superfluous   dye   by 

washing  in  water  or  alcohol  {\  263- 
264),  2-5  minutes. 

16.  Staining  with,  a  general  dye  (§  264), 

15-30  seconds. 

17.  Washing   with    water   or  alcohol 

($  263-264),  1  to  2  minutes. 

18.  Dehydrating  the  sections  in  95%  al- 

cohol (I  266),     5  min.  to  24  hours. 

19.  Clearing  the  sections  (?  266),  5  min. 

to  24  hours. 

20.  Draining  the  sections,     1-2  minutes. 

21.  Mounting  in  Canada  balsam  (\  266), 

1-2  minutes. 

22.  Sealing   the   cover-glass   (\  23S),     ^ 

minutes. 

23.  Labeling  the  preparation  (?  291    . 

minutes. 

24.  Cataloging  the  preparation   {\  294), 

5-10  minutes. 


§266.   Mounting  in  Balsam. — After  the  sections  are  stained  they 
must  be  dehydrated  and  cleared  before  mounting  in  balsam.     For  the 

dehydration  the  slide  is  plunged  into  a  jar  of  95$   alcohol.       For  d 


1 64  PARAFFIN  SECTIONING.  [CH.   VII. 

ing  after  the  dehydration  the  slide  is  drained  of  alcohol  and  put  down 
flat  and  the  clearer  poured  on,  or  the  whole  slide  is  immersed  in  a  jar 
of  clearer  (§  303).  Clearing  usually  is  sufficient  in  a  few  minutes  ;  a 
stay  of  an  hour  or  even  over  night  does  not  injure  most  sections. 

In  mounting  in  balsam  the  clearer  is  drained  away  by  standing  the 
slide  nearly  vertically  on  some  blotting  paper,  or  by  using  the  waste 
bowl  and  standing  it  up  in  the  little  funnel  (Fig.  134).  Then  the  bal- 
sam is  put  on  the  sections  or  spread  on  the  cover-glass  and  that  placed 
over  the  sections. 

For  cataloging  and  labeling,  see  §  291-295. 


Fig.  137.  Small  spirit  lamp  modified  into  a  balsam 
bottle,  or  a  glycerin  or  glycerin-jelly  bottle,  or  a  bottle 
for  homogeneous  immersion  liquid.  For  all  of  these 
purposes  it  should  contain  a  glass  rod  as  shozvn  in  the 
figure.  By  adding  a  small  brush,  it  atiszvers  well  for 
a  shellac  bottle  also. 


\  267.  The  Collodion  Method  with  Alcohol. — A  good  method  of  procedure  for 
making  collodion  sections  is  to  proceed  exactly  as  described  including  \  257,  and 
then  instead  of  hardening  the  collodion  in  chloroform  and  clarifier,  it  is  hardened 
in  82%  alcohol  for  a  day  or  two  before  sectioning.  In  sectioning,  the  knife  and 
tissue  are  kept  wet  with  82%  alcohol  and  the  sections  are  dehydrated  with  95% 
alcohol  and  then  fastened  to  the  slide  with  ether  alone  or  with  ether-alcohol. 
The  staining  and  mounting  ($  263-266)  are  as  described.  One  may  preserve  the 
tissue  after  imbedding  for  a  long  time  in  the  82%  alcohol  before  sectioning,  or  for 
successive  sections.  While  this  method  appears  somewhat  simpler,  the  results  are 
not  so  satisfactory  as  by  the  oil-method  given  above. 

THE   PARAFFIN    METHOD. 

§  268.  As  with  the  collodion  method,  the  tissues  are  first  properly 
fixed  and  hardened  and  then  entirely  filled  with  the  imbedding  mass, 
but  unlike  collodion  the  mass  must  be  entirely  removed  before  the  sec- 
tions are  finally  mounted.  The  tissue  thus  imbedded  and  infiltrated  is 
like  a  homogeneous  mass  and  sections  may  be  cut  of  extreme  thinness. 

§  269.   Harden  perfectly    fresh    tissue    in   picric- alcohol   (§  315) 


CHAP.  /'//.]  PARAFFIN  SECTIONING 

from  one  to  three  days.       Any  good  method  for  fixing  ;in<l  hardening 
the  elements  may  be  used.     One  must  observe  in  each  case,  howev< 
the  special  conditions  necessary  for  each  method.     The  time  might 
longer  or  shorter  thau  for  the  picric-alcohol.     See  Lee,  the  Microtomists' 
Wide-  Mecum. 

If  picric-alcohol  is  used,  pom  it  off  aft  r  the  propel  time  for  fixing 
has  elapsed,  and  add  67'/  alcohol.  Leave  this  on  the  tissue  from  one 
to  three  days,  and  if  it  becomes  very  yellow  it  is  well  to  change  it  two 
or  three  times.  After  two  or  three  days  pour  off  the  67$  alcohol  and 
add  82'/.  The  tissue  should  remain  in  this  one  or  two  days,  and  it 
may  remain  indefinitely. 

In  case  the  alcohol  becomes  much  yellowed,  it  should  be  changed. 

\  270.  Dehydration  and  Preparation  for  Imbedding1 — Prom  the  pieces  of 
tissue  fixed  and  hardened  in  any  approved  manner,  cut  pieces  5  to  [o  millimeti 
long  and  2  to  3  millimeters  in  breadth.  Place  one  or  two  pieces  in  a  shell  vial 
(Fig.  132)  and  add  95'^  alcohol.  Change  the  alcohol  after  two  or  three  hours, 
and  within  6  to  24  hours,  depending  on  the  size  of  the  piece  to  be  dehydrated,  the 
dehydration  will  be  completed.  The  secret  of  success  is  the  use  of  plenty  of 
alcohol  and  sufficient  time.  Absolute  alcohol  for  the  second  change  would  act 
more  promptly  and  efficiently,  but  if  plenty  of  95^  is  used  one  will  succeed,  unless 
the  day,  or  the  climate  in  general,  is  too  damp. 

I  If  one  is  studying  organs,  then  the  whole  organ  may  need  to  be  prepared  for 
imbedding,  but  for  the  minute  structure  small  pieces  are  preferable,  as  thinner 
sections  may  be  made.  1 


> 


1.  Displacing  the  Alcohol  and  Clearing  Tissues  with  Thickened 
Cedar-wood  Oil  and  Infiltrating  with  Paraffin. — 1  Lee,  p.  66.  Neelson  and 
Schiefferdecker,  Arch,  fiir  Anat.  und  Physiol.,  [882,  p.  206.1  When  the  tissue  is 
dehydrated  it  is  removed  to  a  vial  of  thickened  cedar-wood  oil.  When  the  alcohol 
used  for  dehydration  is  displaced  by  the  oil,  the  tissue  will  look  clear  and  trans- 
lucent. This  requires  2  to  24  hours.  It  is  hastened  by  warmth.  It  is  then 
removed  from  the  cedar-wood  oil,  drained,  and  placed  in  pure,  melted  paraffin, 
and  this  is  then  put  into  m  paraffin  oven  and  left  from  2  to  24  hours.  It  is  then 
imbedded  for  sectioning. 

Paraffin  for  infiltrating  lias  usually  a  somewhat  lower  melting  point  than  that 
for  imbedding.  Equal  parts  of  paraffin  of  (3  C.  and  54  C,  answer  well.  For 
imbedding,  the  paraffin  must  be  of  a  melting  point  which  will  give  good  ribbons 
in  the  temperature  of  the  room  where  the  sectioning  is  to  In-  done.  In  a  room  oi 
19  to  20  C.  a  mixture  of  1  part  43  C.  paraffin  with  two  parts  of  54  C.  usually  answers 

well. 

\  272.  Imbedding  in  Paraffin. — Make  a  small  paper  1><>x,  till  it  about  half  full 
of  pure  melted  paraffin  of  tin-  proper  melting  point  I  see  .'71  .  ami  then  remove  the 
tissue  from  the  infiltrating  oven,  place  it  in  one  end  of  tin-  paper  box  ami  arrange 
it  so  that  sections  may  be  made  in  any  desired  direction.  A.s  soon  as  the  paraffin 
has  solidified  on  tin-  surface,  place  tin-  box  in  cold  water,  on  ice  .•!  in  snow  to 
the  paraffin  quickly,  and  thus  avoid  ^1  ac<  s  around  the  tissue, 

\  273.  Cutting  the  Sections.     After  the  imbedding  mass  is  well  cooled,  ren 
the  paper  Imx  and  trim  the  end  containing  tin-  tissue  in  a  pyramidal  form       Clamp 


1 66  PARAFFIN  SECTIONING.  [CHAP.  VII 

the  block  of  paraffin  in  the  holder  of  the  microtome  so  that  the  tissue  will  be  at 
tin-  proper  level  for  cutting.  If  a  ribbon  microtome  is  used,  heat  the  holder  and 
melt  tin-  end  of  tin-  block  upon  it.  Cool  and  place  the  holder  in  its  place  in  the 
microtome,  Us(  a  very  sharp,  dry  razor  for  cutting  the  sections.  The  sections 
art-  made  with  a  rapid,  straight  cut  as  in  planing.  Do  not  try  to  section  with  a 
drawing  out  as  in  collodion  sectioning.  If  the  temperature  of  the  room  is  right 
for  the  paraffin  used,  the  sections  will  remain  flat,  and  if  the  opposite  sides  of  the 
block  are  parallel,  and  one  edge  strikes  the  knife  squarely,  the  sections  will 
adhere  and  thus  make  a  ribbon.  If  the  room  is  too  cold  for  the  paraffin  the  sec- 
tii  us  will  roll.     If  it  is  too  warm  the  sections  will  be  imperfect. 

Remember  the  sections  must  be  very  thin,  from  3/*  to  15^1  to  show  fine  structural 
details  to  good  advantage. 

74.  Extending  the  Sections  with  Warm  Water. — raraffin  sections  are 
liable  to  be  very  finely  wrinkled.  These  wrinkles  in  the  sections  often  obscure  the 
structure.  To  remove  them,  the  ribbons  or  separate  sections  are  placed  on  cold 
water  in  a  dish  like  a  waste  jar  (Fig.  135).  Then  hot  water  is  slowdy  added  till 
the  sections  extend.  This  removes  the  folds.  When  the  water  has  cooled,  the 
ribbons  are  cut  into  proper  lengths  with  scissors,  and  the  pieces  transferred  to 
albumenized  slides. 

:  274a.  Extending  Sections  on  the  Slide. — Instead  of  placing  the  sections 
on  water  in  a  dish,  the  sections  ma}^  be  put  directly  upon  slides.  To  extend  them, 
add  sufficient  water  so  that  they  will  float.  Warm  the  slide  carefully  until  they 
straighten,  pour  off  the  water,  and  allow  the  slides  to  stand  for  several  hours 
until  all  the  water  has  evaporated.  The  sections  adhere  firmly  to  the  slide  and 
are  in  optical  contact  with  it,  as  shown  by  the  shiny  appearance  when  all  the  water 
has  evaporated. 

2  275.  Fastening  the  Sections  to  the  Slide. — To  fasten  the  sections  firmly  to 
the  slide,  coat  the  slide  with  albumen  fixative  ($  297)  as  follows :  Put  a  minute 
drop  of  the  albumen  on  the  center  of  a  slide  and  with  a  clean  finger  spread  the 
albumen  over  the  slide,  wiping  off  all  that  is  possible.  Finally  beatortap  the  slide 
with  the  end  of  the  finger.  This  will  make  a  very  thin  (it  cannot  be  too  thin)  and 
even  layer.  Place  the  sections  in  position  and  allow  them  to  remain  until  the 
water  has  all  evaporated.  It  is  well  to  leave  them  over  night.  After  the  water 
has  evaporated,  coat  the  sections  with  3^ths$  collodion,  using  a  delicate  brush  for 
the  purpose.  Allow  the  collodion  to  dry  for  a  minute  or  two,  then  put  the  slides 
in  benzin  or  xylene  to  dissolve  the  paraffin  (see  \  276).  If  the  sections  are  not 
extended  on  water,  they  may  be  put  directly  on  the  albumenized  slides,  pressed 
down  with  the  finger  and  coated  with  collodion.  This  is  much  more  rapid,  but 
does  not  get  rid  of  the  fine  folds.     See  also  the  preface  for  albumenizing  slides. 

\  276.  Removing  the  Paraffin. — Immerse  the  slide  in  a  vessel  of  xylene  or 
benzin.  This  will  dissolve  the  paraffin.  An  hour  will  usually  suffice.  One  can 
hasten  the  solution  of  the  paraffin  by  moving  the  slide  in  the  solvent.  In  this  way 
it  may  be  dissolved  in  5  to  10  minutes,  or  even  less.  It  will  do  no  harm  to  leave 
the  slide  in  the  benzin  or  xylene  over  night.  Two  or  three  days  even  might  not 
do  any  harm,  but  it  is  usually  better  to  proceed  at  once  to  the  other  operations. 

\  277.  Removing  the  Xylene  or  Benzin — From  the  xylene  or  benzin  plunge 
the  slide  bearing  the  sections  into  a  jar  of  95$  alcohol,  and  leave  it  for  a  few 
minutes,  or  move  it  around  in  the  alcohol  for  half  a  minute  or  so. 

\  278.  Staining  the  Sections  with  an  Alcoholic  Dye. — With  an  alcoholic 


CHAP.  17/.]  PARAFFIN  SECTIONING 

stain  like  hydrochloric  acid  carmine,  remove  the  slide  from  the  alcohol,  and  add 
the  stain  directly  after  draining  the  slide.     Do  no!  allow  the  stain  to  become  di 
for  that  would  injure  the  tissue.     Wash  away   the  stain  with  67$  alcohol,  then 
dehydrate  with  97$  alcohol,  clear  and  mount  in  balsam  as  described  below. 

\  270.  Staining  with  an  Aqueous  Dye.-  Wash   away  thi  ilcohol   from 

the  slide  bearing  the  sections  by  plunging  it  into  a  jar  ot"  water  and  moving  il 
around  a  moment.     Then  add  the  stain  to  the  sections  with  a  pipette,  or  imm< 

tin-    slide    in    a   jar   of   tin-    stain,  and    allow    the    stain    to  act  from  5  to  to  mini.' 
Wash  thoroughly  with  water. 

\  280.  Staining  with  a  General  Dye — Counterstaining. —  If  it  is  desired  to 
give  a  general  stain  after  the  nuclear  dye  1  \  279),  carmine  stained  preparations  maj 
be  tinted  with  picric-alcohol  for  half  a  minute  or  more  (§  315),  and  the  hematoxylin 
stained  specimens  with  eosin  1  \  305  1.  It  usually  takes  less  than  a  minute  for  this. 
Wash  away  the  couutcrstain  with  water. 

£  280a.  Counterstaining  with  Picro-fuchsin.—  For  a  general  dye  t->  use  with 
hematoxylin,  eosin  is  good,  but  to  differentiate  the  tissues  more  completely, 
especially  connective  tissue,  which  is  present  in  practically  every  section  made,  it 
is  better  to  use  Van  Gieson's  mixture'  of  picric  acid  and  acid  fuehsin.  1  Picric  acid, 
saturated  aqueous  solution  75  CC,  water  25  CC.  1  ',  aqueous  solution  of  acid  fuehsin, 
IO  cc.)  Sections  are  first  strongly  stained  with  hematoxylin,  well  washed  with 
water  and  then  stained  3  seconds  to  15  minutes  in  the  picro-fuchsin.  They  are 
then  washed  in  distilled  water  or  in  tap  water,  to  which  has  been  added  a  drop  or 
two  of  glacial  acetic  acid  to  100  cc.  of  the  water.  They  are  then  dehydrated, 
cleared  and  mounted  in  acid  balsam,  that  is  in  balsam  which  has  not  been  neutral- 
ized 1  \  300).  If  glycerin  or  glycerin  jelly  is  used  as  a  mounting  medium  it  should 
be  slightly  acid.  Unlecs  the  mounting  medium  is  slightly  acid,  the  red  of  the  acid 
fuehsin  soon  fades.  In  some  cases  less  acid  fuehsin  should  hi'  used,  and  in  some  1 
greater  amount.  Ac-id  fuehsin  alone  without  the  picric  acid  is  also  good  for  a 
counterstain.  The  picro-fuchsin  is  a  very  valuable  differential  stain  and  combined 
in  different  proportions  with  picric  acid  will  give  great  assistance  in  almost  every 
case.  It  does  not  seem  to  be  a  very  permanent  stain.  See  Freeborn,  Trans.  N. 
V.  Path.  SoC,  (893,  p.  73.  Also  studies  from  the  department  of  pathology  of  the 
College  of  Physicians  and  Surgeons,  Columbia  University,  N.  V.,  [894    iv 

S  2S1 .  Dehydration  of  the  Stained  Sections. — Place  the  slide  with  the  stained 
sections  in  a  jar  of  95^  alcohol  and  leave  it  a  few  minutes,  or  wave  it  around  in 
the  alcohol  for  half  a  minute  or  so. 

\  2S2.  Clearing  the  Sections.  —  Drain  off  the  alcohol,  and  place  tin-  slide  in  a 
jar  of  clearer  303,  A  or  B)  or  put  a  drop  or  two  of  clearer  on  the  sections.  The 
clearing  is  usually  accomplished  in  two  or  three  minutes. 

§  283.  Mounting  in  Balsam. — For  this  the  clearer  is  drained  from  the   slide, 
and  wiped  away  with  lens  or  blotting  paper,  cloth,   etc.     The  balsam  is  then  put 
upon  the  sections  and   the  cover  added,  or  a  cover-glass  is  Spread  with    the  balsam 
and   put   over  the   sections.       I  If  the   sections   show  a  whitish   appearand    and 
opaque  they  wire  not  sufficiently  dehydrated. 


i68 


/'.  /A'.  IFFIN  S/u  'TIONING. 


\CH.    VII 


ORDER     OF     PROCEDURE     IN     MAKING     MICROSCOPICAL     PREPARATIONS     BY     THE 

PARAFFIN     METHOD. 

284.  It  will  be  seen  from  this  tabic  and  from  sections  268  to  283  that  it  re- 
quires  from  5  to  7  days  to  get  a  microscopical  preparation  by  the  paraffin  method 
if  one  starts  with  a  fresh  tissue.  Depending  on  the  method  of  fixing  and  harden- 
ing, the  time  may  be  much  greater.  Unless  mush  time  is  lost  in  waiting  one  must 
plan  ahead  in  histological  work. 


1.   Fixing  and  hardening  the  tissue  or 

organ  ■     269),   |.  days  or  more. 
j.   Dehydrating  the  object  to  he  cut  in 

95V    or  stronger  alcohol   (  ;-  270), 
1  to  24  hours. 
j.    Displacing  the  alcohol  and  clearing 
tissues  with  cedar-wood  oil.      (See 
\  27  1  ),  2  to  24  hours. 

4.  Infiltrating    the   tissue  with  paraffin 

in  the  paraffin  oven   (|  271  ),   2  to 
24  hours. 

5.  Imbedding  in    paraffin    1  \   272),   10 

minutes. 

6.  Cutting  the  sections  1  \  275  1,  10  min- 

utes. 

7.  Extending  the  sections  with  warm 

water.      (  .See  \  274,  274a.  ) 

8.  Fastening   the    sections    to    a   slide 

(  \  275  ),  5  minutes  to  24  hours. 

9.  Removing  the   paraffin    \\    276),   10 

minutes  to  24  hours. 
10.   Removing     the    xylene    or     benzin 
277)- 


11. 
12. 


14. 

15- 
16. 


18. 
r9. 
20. 
21. 


Washing  with  water,  note,  p.  162. 
Staining     with     an      aqueous     dye 

{\  279),  2  minutes  to  24  hours. 
Washing  away  the  superfluous  stain 

with  water  (  \  279). 
Staining  with  a  general  dye  [\  280- 

280a),  10  seconds  to  10  minutes. 
Washing    the    sections   with    water 

(J  28o-28oa). 

Dehydrating  the  stained  sections  in 
95%  alcohol  (?  281),  3  minutes  to 
24  hours. 

Clearing  the  sections  (i  282),  2  min- 
utes to  24  hours. 

Mounting  in  Balsam  (?  283),  1  to  5 
minutes. 

Sealing   the   cover-glass    [\    23S),    2 

minutes. 
Labeling  the  preparation   (2  292),  2 

minutes. 
Cataloging  the  preparation   ( \  294), 

5  to  10  minutes. 


SERIAL    SECTIONS. 


S  285.  In  histological  studies  it  is  frequently  of  the  greatest  advant- 
age to  have  the  sections  in  serial  order,  then  an  obscure  feature  in  one 
section  is  frequently  made  clear  by  the  following  or  preceding  sections. 
While  serial  sections  are  very  desirable  in  histological  study,  they  are 
absolutely  necessary  for  the  solution  of  morphological  problems  pre- 
sented in  complex  organs  like  the  brain,  in  embryos  and  in  minute 
animals  where  gross  dissection   is  impossible. 

ij  286.  Arrangement  of  Tissues  for  Sections  in  Histology  . — 
They  should  be  so  arranged  that  the  exact  relations  of  each  part  to  the 
organ  can  be  readily  determined.  For  example,  an  organ  like  the  in- 
testine, a  muscle  or  a  nerve,  should  be  so  airanged  that  exact  transec- 


<//.   17/.]  SERIAL  SECTIONS. 

tious  or  longisections  can  be  made.  Organs  like  the  liver  and  other 
glands,  the  skin,  etc.,  should  be  so  arranged  that  sections  parallel  with 
the  surface  or  at  right  angles  to  it,  I  surface  or  vertical  sections  may  be 
made.     Oblique  sections  are  often  very  puzzling. 

With  cylindrical  objects,  especially  botanical  specimens,  one  niaj  cut 
tangential  sections,  /.  e. ,  sections  at  right  angles  to  a  radius,  «.r  parallel 
with  the  radii   (radial  sections),   or  transections,   /.  e.,  sections  acr< 
the  long  axis. 

.^  287.  Arrangement  of  Serial   Sections. — The   numerical    01 
may  be  very  conveniently  like  the  words  on  a  printed  page,  from  the 
upper  left  hand  corner  and  extending  from  left  to  right,  top  to  bottom 

(Fig-  135). 

The  position  of  the  variotis  aspects  of  the  sections  should  be  in  gen- 
eral such  that  when  they  are  under  the  compound  microscope  the  rights 
and  lefts  will  correspond  with  those  of  the  observer.  This  may  be  ac- 
complished as  follows  for  sections  made  in  the  three  cardinal  sectional 
planes,  Transections ',  Frontal  Sections,  Sagittal  Sections  : 

(A)  Transections,  i.  c,  sections  across  the  long  axis  of  the  embryo 
or  animal  dividing  it  into  equal  or  unequal  cephalic  and  caudal   parts. 

I  a  l  In  accordance  with  the  generally  approved  method  of  numbering 
serial  parts  in  anatomy,  the  most  cephalic  section  should  be  first  No. 
1  of  Fig.  135). 

(b)  The  caudal  aspect  of  the  section  should  face  upward  toward  the 
cover-glass,  the  cephalic  aspect  being  next  the  slide. 

(c)  The  ventral  aspect  should  face  toward  the  upper  edge  of  the 
slide  (Fig.  135). 

This  arrangement  may  be  easily  accomplished  in  transections  in  two 
ways  :  (  1  )  The  embryo  or  animal  is  imbedded  in  such  a  way  that  the 
sectioning  shall  begin  at  the  cephalic  end.  In  this  case  the  first  section 
is  placed  in  the  upper  left  hand  corner  of  the  slide  1  Xo.  1  of  Fig.  i,;: 
but  it  must  be  turned  over  so  that  the  caudal  aspect  shall  face  up. 
The  ventral  aspect  must  be  made  to  look  toward  the  upper  edge  of  the 
slide,  then  under  the  compound  microscope  the  dorsal  side  will  appear 
toward  the  upper  edge  of  the  slide  and  the  right  and  left  correspond 
with  the  observer. 

2  |  The  embryo  or  animal  is  imbedded  so  that  the  sectioning  begins 
at  the  caudal  end,  then  the  sections  are  not  turned  over,  as  thej  are  al- 
ready caudal  face  up,  bnt  they  must  be  put  on  the  slide  in  reverse  order, 
i.  c,  the  first  section  made  is  put  in  the  lower  right  hand  corner  N 
10  of  Fig.  135).  In  this  way  the  most  cephalic  section  will  be  number 
one  as  before.  As  in  the  previous  case  the  ventral  side  of  the  section 
should  be  toward  the  upper  edge  of  the  slide-  1  Pig.  [35  . 


170  SERIAL  SECTIONS.  [CH.    VII. 

B  l  Frontal  sections,  i.  e.,  sections  lengthwise  of  the  embryo  or  ani- 
mal and  from  right  to  left  (dextral  and  sinistral),  so  that  it  is  divided 
into  equal  or  unequal  dorsal  and  ventral  parts. 

The  embryo  is  so  imbedded  and  arranged  in  the  microtome  that  the 
dorsal  part  is  cut  first.  The  first  section  is  then  placed  in  the  upper 
left  hand  corner  (No.  i,  Fig.  135)  dorsal  side  up,  and  the  cephalic  end 
toward  the  lower  edge  of  the  slide.  The  microscopic  image  will  then 
appear  with  right  and  left  as  in  the  observer. 

C)  Sagittal  sections,  i.  <?.,  sections  lengthwise  of  the  embryo  or  ani- 
mal, and  from  the  ventral  to  the  dorsal  side,  thus  dividing  it  into  equal 
or  unequal  right  and  left  parts.  For  these  the  left  side  of  the  embryo  is 
placed  up  so  that  it  is  cut  first.  The  first  section  is  placed  in  the  upper 
left  hand  corner  of  the  slide,  the  left  aspect  facing  away  from  the  slide 
and  the  head  to  the  right  end,  the  ventral  side  toward  the  upper  edge 
of  the  slide.  Under  the  microscope  the  dorsal  side  will  then  appear 
toward  the  upper  edge  of  the  slide  and  the  head  to  the  left. 

£  2S8.  For  serial  sections  with  collodion  imbedded  objects  it  is  a 
great  advantage  to  have  the  imbedding  mass  unsymmetrically  trimmed, 
so  that  if  a  section  is  accidentally  turned  over  it  may  be  easily  noticed 
and  rectified. 

Furthermore  it  is  imperatively  necessary  that  the  object  be  so  im- 
bedded that  the  cardinal  aspects,  dextral  and  sinistral,  dorsal  and  ven- 
tral, cephalic  and  caudal,  shall  be  known  with  certainty. 

UPPER   EDGE   OF   SLIDE. 


Series  No.  75.                  Cover  .15  mm. 
Slide  No.  1. 

c 
z 

w 

1 

2 

3 

4 

5 

Transections  of  a  Diemyctylus 
Embryo. 

Eh 

Sections  1-10. 

W 

6 

7 

8 

9 

10 

Total  thickness  of  Sections,  1  mm. 
May  20,  1S92. 

Fig.  135. — Labeled  Slide  of  Serial  Sections. 

^  289.  Thickness  of  Cover. Glass  and  of  Serial  Sections. — It  is 
a  great  advantage  to  use  very  thin  cover-glasses  (TW  to  x\%  mm.)  for 
serial  sections,  then  the  cover  will  not  prevent  the  use  of  high  powers. 
When  the  ordinary  slides  (25  x  76  mm.,  1  x  3  inch)  are  used  cover- 
glasses  23  X  55  mm.  may  be  advantageously  employed. 

The  combined  thickness  of  the  sections  on  a  slide  is  easily  determined 
by  noting  carefully  the  position  of  the  microtome  screw  at  the  first  and 
last  sections,  and  measuring  the  elevation.     Then   if  the  sections  are 


CH.   V/I.]  LABELING  AND  CATALOGING.  171 

uniform  the  thickness  of  each  may   be  easily   found.      The  avei 
thickness  may  be  easily  determined  in  any  case. 

§290.   Labeling  Serial  Sections. — The  label  of  a  slide  on  which 
rial  sections  are  mounted  should  contain  at  least  the  following  :  (1)  The 

number  of  the  series  ;   (  2  )  The  number  of  the  slide  in  the  scries  I  if  the 
series  required  more  than  one  slide)  ;   (3)    Kind  of  sections  i  transections, 

etc.)  and  the  name  of  the  object  from  which  derived  ;  (4)  The  number 

of  the  first  and  last  section  on  the  slide  ;   (5)  The  total  thickness  of  all 
the  sections  on  the  slide  ;   (6)  The  date  of  the  series. 

LABELING,  CATALOGING  AND  STORING  MICROSCOPICAL  PREPARATIONS. 

§  291.  Every  person  possessing  a  microscopical  preparation  is  inter- 
ested in  its  proper  management  ;  but  it  is  especially  to  the  teacher  and 
the  investigator  that  the  labeling,  cataloging  and  storing  of  microscop- 
ical preparations  are  of  importance.  "To  the  investigator,  his  speci- 
mens are  the  most  precious  of  his  possessions,  for  they  contain  the  facts 
which  he  tries  to  interpret,  and  they  remain  the  same  while  his  knowl- 
edge, and  hence  his  power  of  interpretation,  increase.  They  thus  form 
the  basis  of  further  or  more  correct  knowledge  ;  but  in  order  to  be 
safe  guides  for  the  student,  teacher,  or  investigator,  it  seems  to  the  wri- 
ter that  ever}'  preparation  should  possess  two  things  :  viz.,  a  label  and  a 
catalog  or  history.  This  catalog  should  indicate  all  that  is  known  of  a 
specimen  at  the  time  of  its  preparation,  and  all  of  the  processes  by 
which  it  is  treated.  It  is  only  by  the  possession  of  such  a  complete 
knowledge  of  the  entire  history  of  a  preparation  that  one  is  able  to 
judge  with  certainty  of  the  comparative  excellence  of  methods,  and 
thus  to  discard  or  improve  those  which  are  defective.  The  teacher,  as 
well  as  the  investigator,  should  have  this  information  in  an  accessible 
form,  so  that  not  only  he,  but  his  students  can  obtain  at  any  time, 
all  necessary  information  concerning  the  preparations  which  serve  him 
as  illustrations  and  them  as  examples. ' ' 

§  292.  Labeling  Ordinary  Microscopical  Preparations. — The 
label  should  possess  at  least  the  following  information  (see  §  290  for 
serial  sections)  : 

KXAMl'LK. 


No-  -,75-       feci  £» 


(  1)  The  number  of  the  preparations,  the 

thickness  of  the  cover-glass  and 

of  the  sections  under  it. 
, N  >,.,,„.    .  ,  r.t  Stnated     Muscle;     transection    of   the 

(2 )  I  lie  name  and  source  of  the  prepara- 

tion. S  irtoriua  of  the  Cat. 

(3)  The   date   of   the    specimen    (  2   of 

catalog.)  October  15,   1S94. 


I.AIiELING  AND  CATALOGING. 


\_CH.    VII 


$  293.  Cataloging  Preparations. — It  is  believed  from  personal  ex- 
perience, and  from  the  experience  of  others,  that  each  preparation 
I  each  slide  or  each  series )  should  be  accompanied  b}^  a  catalog  contain- 
in-  at  least  the  information  suggested  in  the  following  formula  :  This 
formula  is  very  flexible,  so  that  the  order  may  be  changed,  and  num- 
bers not  applicable  in  a  given  case  may  be  omitted.  With  many  ob- 
jects, especially  embryos  and  small  animals,  the  time  of  fixing  and 
hardening  may  be  months  or  even  years  earlier  than  the  time  of  im- 
bedding. So,  too,  an  object  may  be  sectioned  a  long  time  after  it  was 
imbedded,  and  finally  the  sections  may  not  be  mounted  at  the  time  they 
are  cut.  It  would  be  well  in  such  cases  to  give  the  date  of  fixing  under 
2,  and  under  5,  6  and  8,  the  dates  at  which  the  operations  were  per- 
formed if  they  differ  from  the  original  date  and  from  one  another.  In 
brief,  the  more  that  is  known  about  a  preparation  the  greater  its  value. 


\  294.   General    Formula   for   Catalog- 
ing Microscopical  Preparations  : 


A  Catalog  Card  Written  According  to 
this  Formula  : 

Muscular  Fibers.     Cat. 

C.  15. 
Fibers  20  to  40  //  thick.. 

2.  No.  475.  (Drr.  IX)  Oct.  t,  1891.  S. 
H.  G.,  Preparator. 

3.  Tendinous  and  iiitra-tnuscular  ter- 
minations of  striated  muscular  fibers 
from  the  Sartorius  of  the  cat  (Felis  do- 
mes lica.) 

4.  Cat  eight  months  old,  healthy  and 
well  nourished.     Fasting  and    quiet   for 

4.  The  age  and  condition  of  the  object    12  hours. 

from  which  the  preparation   is  derived.  5.  Musc]e  pinned  on   cork   with   vas. 

Condition    of    rest    or    activity  ;  fasting  eiined  pins  and  placed   in    20  per  cent, 

or  full  fed  at  the  time  of  death.  llitric  acid  immediately   after  death   by 

5.  The     chemical     treatment,  -the  chloroform-     Left  36  hours  in  the  acid  ; 
method  of  fixing,  hardening,  dissociating  teniPerature  20°  C.     In   alum   water  {% 


1.  The  general  name  and  source. 
Thickness  of  cover  glass  and  of  sections. 

2.  The  number  of  the  preparation  and 
the  date  of  obtaining  and  fixing  the 
specimen  ;  the   name  of  the  preparator. 

3.  The  special  name  of  the  prepara- 
tion and  the  common  and  scientific 
name  of  the  object  from  which  it  is  de- 
rived.    Purpose  of  the  preparation. 


etc.,  and  the  time  required. 

6.  The  mechanical  treatment, — im- 
bedded, sectioned,  dissected  with  nee- 
dles, etc.     Date  at  which  done. 

7.  The  staining  agent  or  agents  and 
the  time  required  for  staining. 


8.  Dehydrating  and  clearing  agent, 
mounting  medium,  cement  used  for 
sealing. 


sat.  aq.  sol.)   1  day. 

6.  Fibers  separated  on  the  slide  with 
needles.  Oct.  3. 

7.  Stained  5  minutes  with  Dekfield's 
hematoxylin. 

8.  Dehydrated  with  95%.  alcohol  5 
minutes,  cleared  5  minutes  with  carbol- 
turpentine,  mounted  in  xylene  balsam  ; 
sealed  with  shellac. 


9.  Use  18  mm.  for  the  general  appear- 
ance of  the  fibers,  then  2  or  3  mm.  ob- 
jective for  the  details  of  structure.     Try 

9.  The  objectives   and  other  accesso-    the  micro-polariscope  ($  209). 

ries  (micro-spectroscope,   polarizer,  etc. )         10.  The  nuclei   or   muscle   corpuscles 
for  studying  the  preparation.  are  very  large  and  numerous  ;   many  of 

the   intra-muscular  ends  are    branched. 

10.  Remarks,  including  references  to  See  S.  P.  Gage,  Proc.  Amer.  Micr.  Sci., 
original  papers,  or  to  good  figures  and  1890,  p.  132  ;  Ref.  Hand-book  Med.,  Sci., 
descriptions  in  books.  Vol.  V.,  p.  59 


CH.    I  'II ]  L  ABEL ING  A NL >  CATAL OGINC.  i  7 .; 

\  295  General  Remarks  on  Catalogs  and  Labels.  -It  is  especially  desirable  that 
labels  and  catalogs  shall  he  written  with  some  imperishable  ink.  Some  form  "I 
waterproof  carbon  ink  is  the  most  available  and  satisfactory.  The  water-proof 
India  ink,  or  the  engrossing  carbon  ink  of  Iliggins,  answers  very  well.  As  pur- 
chased, the  last  is  too  thick  for  ordinary  writing  and  should  be  diluted  with  one- 
third  its  volume  of  water  and  a  few  drops  of  strong  ammonia  added. 

If  one  has  a  writing  diamond  it  is  a  good  plan  to  write  a  label  with  it  on  one  end 
of  the  slide.      It  is  best  to  have  the  paper  label  also,  as  it  can  be  more  easily  read. 

Cwiinnn|wnwnMnrmpa<iinin«nnmwarwniiMiniiiiin  imw  t  fCffho 
' 1  '11  -      1  •        1  1  11  iinn -•-   -  -  ■-  ■    -  —  *  ■  \31T 

Fig.  136.  Writing  diamond  for  writing  numbers  and  labels  on  glass  slides,  cut- 
ting cover-glasses,  etc.     {Queen  &  Co.). 

The  author  has  found  stiff  cards,  12^x7^  cm.,  like  those  used  for  cataloging 
books  in  public  libraries,  the  most  desirable  form  of  catalog.  A  specimen  that  is 
for  any  cause  discarded  has  its  catalog  card  destroyed.  New  cards  may  then  be 
adde  1  in  alphabetical  order  as  the  preparations  are  made.  In  fact  a  catalog  on 
cards  has  all  the  flexibility  and  advantages  of  the  slip  system  of  notes  (see  Wilder 
,\:  Gage,  p.  45). 

Some  workers  prefer  a  book  catalog.  Very  excellent  book  catalogs  have  been 
devised  by  Ailing  and  by  Ward  (Jour.  Roy.  Micr.  Soc,  18S7,  pp.  173,  34S  ;  Amer. 
Monthly  Micr.  Jour.,  1890,  p.  91  ;  Amer.  Micr.  Soc.  Proc,  1887,  p.  233). 

The  fourth  division  has  been  added  as  there  is  coming  to  be  a  very  strong  belief, 
practically  amounting  to  a  certainty,  that  there  is  a  different  structural  appearance 
in  many  if  not  all  of  the  tissue  elements  depending  upon  the  age  of  the  animal, 
upon  its  condition  of  rest  or  fatigue  ;  and  for  the  cells  of  the  digestive  organs, 
whether  the  animal  is  fasting  or  full  fed.  Indeed  as  physiological  histology  is 
recognized  as  the  only  true  histology,  there  will  be  an  effort  to  determine  exact 
data  concerning  the  animal  from  which  the  tissues  are  derived.  (SeeMinot,  Proc. 
Amer.  Assoc.  Adv.  Science,  189),  pp.  271-2S9  ;  Hodge,  on  nerve  cells  in  rest  and 
fatigue,  Jour.  Morph.,  vol.  VII.  (1892),  pp.  95-168  ;  Jour.  Physiol.,  vol.  XVII., 
pp.  129-134;  Gage,  The  processes  of  life  revealed  by  the  microscope  ;  a  plea  for 
physiological  histology,  Proc.  Amer.  Micr.  Soc,  vol.  XVII.  (  1895),  pp.  3-29  ;  Sci- 
ence, vol.  II.,  Aug.  23,  1895,  pp.  209-218). 

CABINET    FOR    MICROSCOPICAL    PREPARATIONS. 

\  296.  While  it  is  desirable  that  microscopical  preparations  should  be  properly 
labeled  and  cataloged,  it  is  equally  important  that  they  should  be  protected  from 
injury.  During  the  last  few  years  several  forms  of  cabinets  or  slide  holders  have 
been  devised.  Some  are  very  cheap  and  convenient  where  one  has  but  a  lew 
slides.  For  a  laboratory  or  for  a  private  collection  where  the  slides  are  numerous 
the  following  characters  seem  to  the  writer  essential  : 

(]).  The  cabinet  should  allow  the  slides  to  lie  flat,  and  exclude  dust  and  light. 

(2).  Each  slide  or  pair  of  slides  should  be  in  a  separate  compartment     At  each 
end  of  the  compartment  should  be  a  groove  or  bevel,  so  that   upon  depressing 
either  end  of  the  slide  the  other  may  be  easily  grasped  (big.  140).     [tisalsodes 
ble  to  have  the  floor  of  the   compartment   grooved  so  that   the  slide  rests  only   on 
two  edges,  thus  preventing  soiling  the  slide  opposite  the  object. 

(3).  Each  compartment  or  each  space  sufficient  to  contain  one  slide  of  the 
standard  size  should  be  numbered,  preferably  at  each   end.      If  the  compartments 


174 


LABELING  AND  CATALOGING. 


\CH.    VII. 


96 


are  made  of  sufficient  width  to  receive  two  slides,  then  the  double  slides  so  fre- 
quently used  in  mounting  seiial  sections  may  be  put  into  the  cabinet  in  any  place 
desired. 

a  B  Fig.  ij.o. — A — .Part  of  a  cabinet  draiver  seen 

from  above.  In  compartment  No.  96  is  repre- 
sented a  slide  lying  flat.  The  label  of  the 
slide  and  the  number  of  the  compartment  are 
so  placed  that  the  number  of  the  compartment 
may  be  seen  through  the  slide.  The  sealing 
cement  is  removed  at  one  place  to  show  that  in 

O  sealing    the    cover-glass,    the    cement  is  put 

I      „       partly  on   the  cover  and  partly  on  the  slide. 
I  (§229,234). 

B. —  This  represents  a  section  of  the  same 
part  of  the  drawer,  (a)  Slide  resting  as  in 
A.  ATo.  96  The  preparation  is  seen  to  be 
above  a  groove  in  the  floor  of  the  compart- 
ment, (b)  One  end  of  the  slide  is  seen  to  be 
uplifted  by  depressing  the  other  into  the  bevel. 


J'o.96   /SSO 
yfert/e.  ft  ten 
yCat 


70 


Fig.  140. 


(4).  The  drawers  of  the  cabinet  should  be 
entirely  independent,  so  that  any  drawer  may 
be  partly  or  wholly  removed  without  disturb- 
ing any  of  the  others. 

(5).  On  the  front  of  each  drawer  should  be 
the  number  of  the  drawer  in  Roman  numer- 
als, and  the  number  of  the  first  and  last  com- 
partment in  the  drawer  in  Arabic  numerals. 
(Fig.  141). 


Fig.  141. — Cabinet  for  Mi- 
croscopical Specimens,  show- 
ing the  method  of  arrange- 
ment and  of  numbering  the 
drawers  and  indicating  the 
number  of  the  first  a?td  last 
compartment  in  each  draiver. 
It  is  better  to  have  the  slides 
on  which  the  drawers  rest 
someivhat  shorter,  then  the 
drawer  front  may  be  entire 
and  not  notched  as  here 
shown.  ( From  Proc.  Amer. 
Micr.  Soc.  1883). 


CIl.   I'//.]  PREPARATION  OF  REAGENTS.  [75 

REAGENTS    FOB    FIXING,    MOUNTING,    ETC. 

\  297.    Albumen  Fixative  (Mayer's). — This  consists  of  equal  parts  ol  well -beaten 
white  of'egg  and   glycerin.      To  each  50  cc.  of  this  1  gram  of  salicylate  of  B< 
is  added  to  prevent  putrefactive  changes.      Probably   a   small  amount   of  formal- 
dehyde, say  1  cc.  of  the  40%,  to  50  or  100  cc.  of  the  fixative  would  suffice  ;  if  too 
strong  the  albumen  would  be  precipitated.     For  method  of  use  see  \  275. 

#298.  Alcohol  (Ethylic) — Ethyl  alcohol  is  mostly  used  for  histological  pur- 
poses. (A)  Absolute  alcohol  (i.  e.  alcohol  of  99-100%)  is  recommended  for  man) 
purposes,  but  if  plenty  of95%alcohol  is  used  it  answers  every  purpose  in  histology. 

(  B)  S2%  alcohol  made  by  mixing  5  parts  of  95%  alcohol  with  1  part  of  water. 

(C)  67%  alcohol  made  by  mixing  2  parts  of  95%  alcohol  with  1  part  of  water. 

\  299.  Alum  Solution. — l'or  muscle  dissociated  in  nitric  acid  (?  2491  a  saturated 
solution  (i.  e.  a  solution  in  which  the  water  holds  all  the  alum  it  can.  If  one  adds 
an  excess  so  that  there  will  always  he  some  undissolved  alum  in  thi  he  can 

he  sure  the  solution  is  saturated  after  it  has  stood  a  few  days.  An  easy  way  to  get 
a  saturated  solution  is  to  take  500  cc.  of  water  and  add  100  grams  of  alum  and  1. 
the  water  in  an  agate  dish.  All  the  alum  will  he  melted,  hut  on  cooling  a  pari 
will  crystallize  out,  leaving  a  cold  saturated  solution  ).  The  saturated  solution  may 
be  used  but,  if  a  half  saturated  solution  is  employed,  it  will  answer  all  the  pur- 
poses. For  a  half  saturated  solution  take  100  cc.  of  water  and  ico  cc.  of  saturated 
alum  water  and  mix  the  two. 

\  300.  Balsam,  Canada  Balsam,  Balsam  of  Fir;  Xylene  Balsam. — This  is  one 
of  the  oldest  and  most  satisfactory  of  the  resinous  media  used  for  mounting  micro- 
scopical preparations.  Sometimes  it  is  used  in  the  natural  state,  but  experience 
has  shown  that  it  is  better  to  get  rid  of  the  natural  volatile  constituents.  A  con- 
siderable quantity,  half  a  liter  or  more,  of  the  natural  balsam  is  poured  into  shal- 
low plates  in  layers  about  1  or  2  centimeters  thick,  then  the  plates  are  put  in  a 
warm,  dry  place,  on  the  back  of  a  stove  or  on  a  steam  radiator,  and  allowed  to  re- 
main until  the  balsam  may  be  powdered  when  it  is  cold.  This  requires  a  long 
time,  the  time  depending  on  the  temperature  and  the  thickness  of  the  layer  of 
balsam.  By  heating  the  natural  balsam  in  a  tin  or  agate  vessel  over  a  Bun  sen 
burner  or  an  alcohol  lamp  the  time  may  he  greatly  abbreviated.  The  heat  should 
not  be  sufficient  to  boil  the  balsam,  however. 

When  the  volatile  products  have  evaporated,  the  balsam  is  broken  into  small 
pieces  or  powdered  in  a  mortar  and  mixed  with  about  an  equal  volume  of  xylene, 
turpentine  or  chloroform.  It  will  dissolve  in  these,  and  then  should  be  filtered 
through  absorbent  cotton  or  a  filter  paper,  using  a  paper  funnel.*  The  balsam  is 
too  thin  in  this  condition  for  mounting,  but  so  made  for  the  sake  of  filtering  it. 
After  it  is  filtered  it  is  evaporated  slowly  in  an  open  dish  or  a  wide-mouth  bottle  or 
jar  till  it  is  of  a  syrupy  consistency  at  the  ordinary  temperature.  It  is  then  poured 
into  a  bottle  with  a  glass  cap  like  a  spirit  lamp.  For  use  it  is  put  into  .1  small 
spirit  lamp  (Fig.  137). 


*For  filtering  balsam  and  all  resinous  and  gummy  materials,  the  writer  has 
found  a  paper  funnel  the  most  satisfactory.  It  can  be  used  once  and  then  thrown 
away.  Such  a  funnel  may  be  very  easily  made  by  rolling  a  sheet  of  thick  writing 
paper  in  the  form  of  a  cone  and  cementing  the  paper  where  it  overlaps,  or  wind- 
ing a  string  several  times  around  the  lower  part.  Such  a  funnel  is  id  in  one 
of  the  rings  for  holding  funnels. 


1 7  6  /  'R  /•-"/'.  /  R  A  TION  OF  RE  A  GENTS.  [  CH.    1  If. 

The  xylene  is  much  the  bast  substance  to  use  for  thinning  the  balsam.     Such 
xylene  balsam,  as  it  is  then  called,  may  be  used  for  mounting  any  object  suitable 
for  balsam  mounting.     The  dehydration  must  be  very  perfect,  however,  as  xylene 
is  wholly  immiscible  with  water. 

Natural  balsam  is  liable  to  be  slightly  acid.  This  is  of  advantage  for  mounting 
sections  stained  with  carmine  or  injected  with  carmin  gelatin  or  Berlin  blue  gela- 
tin. For  hematoxylin  preparations  and  for  fuchsia  preparations  the  acid  will  cause 
the  color  to  fade.  The  balsam  may  be  neutralized  by  mixing  some  carbonate  of 
soda  with  the  thinned  solution  before  it  is  thickened.  In  a  few  days  all  the  soda 
will  settle  and  the  clear  balsam  above  will  be  neutral  and  may  be  poured  off  and 
thickened.  If  one  mounts  carmine  or  Berlin  blue  preparations  in  the  neutral  bal- 
sam the  blue  will  fade  and  the  carmine  diffuse. 

\  301.  Chloroform  Paraffin. — This  is  made  by  mixing  the  4  parts  of  the  paraffin 
used  for  imbedding  (J  314)  with  1  part  of  chloroform.  This  gives  a  paraffin  which 
melts  at  a  lower  temperature  than  the  pure  paraffin.  If  it  is  kept  warm  the  chloro- 
form evaporates  in  3  to  6  days,  leaving  pure  paraffin. 

\  302.  Clanfler,  Castor-Xylene  Clanfier. — This  is  composed  of  castor  oil  1  part 
and  xylene*  3  parts. 

\  ;<>3.  Clearing  Mixture  (#266,  282).  —  (A).  One  of  the  most  satisfactory  and 
generally  applicable  clearers  is  carbol  turpentine,  made  by  mixing  carbolic  acid 
crystals  {Acidum  carbolicum.  A.  phenicum  crystalizatum)  40  cc.  with  rectified 
oil  of  turpentine  {Oleum  terebinthinae  rectificatum)  6o  cc.  If  the  carbolic  acid 
does  not  dissolve  in  the  turpentine  add  5  cc.  of  95%  alcohol,  or  increase  the  tur- 
pentine, thus  :  carbolic  acid  30  cc,  turpentine  70  cc. 

(B).  Carbol-Xylene,  Clearer. — Vasale  recommends  as  a  clearer,  xylene  75  cc, 
carbolic  acid  (melted  crystals)  25  cc.      It  is  used  in  the  same  way  as  the  preceding. 

\  304  Collodion. — This  is  a  solution  of  soluble  cottouf  or  other  form  of  pyroxy- 
lin in  equal  parts  of  sulphuric  ether  and  95%  alcohol.     Three  solutions  are  used  : 

*The  hydrocarbon  xylene  (C8HI0)  is  called  xylol  in  German.  In  English, 
members  of  the  hydrocarbon  series  have  the  termination  "ene"  while  members 
of  the  alcohol  series  terminate  in   "  ol." 

ffhe  substance  used  in  preparing  collodion  goes  by  various  names,  soluble  cot- 
ton or  collodion  cotton  is  perhaps  best.  This  is  cellulose  nitrate,  and  consists  of  a 
mixture  of  cellulose  tetranitrate  C12  Hl6  (N03)406,  and  cellulose  pentanitrate,  Cia 
H„  (N03).  05.  Besides  the  names  soluble  and  collodion  cotton,  it  is  called  gun 
cotton  and  pyroxylin.  Pyroxylin  is  the  more  general  term  and  includes  several 
of  the  cellulose  nitrates.  Celloidin  is  a  patent  preparation  of  pyroxylin,  more  ex- 
pensive than  soluble  cotton,  but  in  no  way  superior  to  it  for  imbedding. 

Soluble  cotton  should  be  kept  in  the  dark  to  avoid  decomposition.  After  it  is  in 
solution  this  decomposition  is  not  so  liable  to  occur.  The  decomposition  of  the  dry 
cotton  gives  rise  to  nitrous  acid,  and  hence  it  is  best  to  keep  it  in  a  box  loosely 
covered  so  that  the  nitrous  acid  may  escape. 

Cellulose  nitrate  is  explosive  under  concussion  and  when  heated  to  1500  centi- 
grade. In  the  air,  the  loose  soluble  cotton  burns  without  explosion.  It  is  said  not 
to  injure  the  hand  if  held  upon  it  during  ignition  and  that  it  does  not  fire  gun- 
powder if  burned  upon  it.  So  far  as  known  to  the  writer,  no  accident  has  ever 
occurred  from  the  use  of  soluble  cotton  for  microscopical  purpores.  I  wish  to  ex- 
press my  thanks  to  Professor  W.  R.  Orndorff,  organic  chemist  in  Cornell  Univer- 
sity, for  the  above  information.  Proc  Amer.  Micr.  Soc,  vol.  XVII  (1895,),  pp. 
361-370. 


i  "//.    /'//.]  PREPARATION  OF  REAGENTS.  i 


/  / 


\  i.  6%  or  tliick  collodion.     It  is  made  by  mixing  50  cc.  of  sulphuric  ether  and 
50  cc.  of  95%  alcohol  and  adding  6  grams  of  soluble  cotton,     [f  this  is  shaken 
peatedly  the  solution  will  be  complete  in  a  day  or  two. 

B).   iyi%  or  thin  collodion.     To  prepare  this   1 «,   grams  of  soluble  cotton  are 
added  to  100  cc.  of  ether  alcohol  1      v 

U  %  collodion  or  cementing  collodion.     To  prepare  ths  of  a  gram  of 

soluble  cotton  is  adde<l  t<>  10  >  cc.  of  ether-alcohol. 

As  both  ether  and  alcohol  are  very  volatile  it  is  necessary  to  keep  the  bottles 
containing  them  well  corked. 

J  305.  Eosin. —  This  is  used  mostly  as  a  contrast  stain  with  hematoxylin,  which 
is  an  almost  purely  nuclear  stain.  It  serves  to  stain  the  cell-body,  ground  sub- 
stance, etc.,  which  would  be  too  transparent  and  invisible  with  hematoxylin  alone 
If  eosin  is  used  alone  it  gives  a  decided  color  to  the  tissue  and  thus  aids  in  it- 
study  \\  [35).  Eosin  is  used  in  alcoholic  and  in  aqueous  solutions.  A  very  satis- 
factory stain  is  made  as  follows  :  50  cc.  of  water  and  50  cc.  of  95%  alcohol  are 
mixed  and  r-ioth  of  a  gram  of  dry  eosin  added. 

The  eosin  is  used  after  the   hematoxylin  in    most  cases  1  \  280),  and,  as  it  is  in 
alcoholic  solution,  it  may   be  washed  off  with  95%  alcohol  if  the  object  is  to  be 
mounted  in  balsam.     If  it  is  to  be  mounted  in  glycerin  or  glycerin  jelly,  the  ex< 
of  eosin  should  be  washed  away  with  distilled  water. 

\  306.  Ether,  Ether-Alcohol. — Sulphuric  ether  is  meant  when  ether  is  men- 
tioned in  this  book.  For  the  ether-alcohol  mentioned  in  \  254,  304,  etc.,  a  mixture 
of  equal  volumes  of  sulphuric  ether  and  95%  alcohol  is  meant. 

\  307.  Warrant's  Solution. —  Take  25  grains  of  clean,  dry,  gum  arabic ;  25  cc.  of 
a  saturated  aqueous  solution  of  arsenious  acid  ;  25  cc.  of  glycerin.  The  gum  ara- 
bic is  soaked  for  several  days  in  the  arsenic  water,  then  the  glycerin  is  added  and 
carefully  mixed  with  the  dissolved  or  softened  gum  arabic. 

This  medium  retains  air  bubbles  with  great  tenacity.  It  is  much  easier  to  avoid 
than  to  get  rid  of  them  in  mounting. 

\  308.  Formaldehyde  Diasociator.  — This  is  composed  of  5  cc.  of  a  \o%  solution 
of  formaldehyde  in  995  cc.  of  water,  to  which  6  grains  of  common  table  salt 
(sodium  chlorid  \  have  been  added.  That  is,  it  is  a  ,J,,%  solution  of  formalde- 
hyde in  normal  salt  solution  (2  313)-  Formaldehyde  as  bought  in  the  market  is  a 
40%  solution  in  water,  and  is  called  formol,  formalin,  formalose  and  formal,  the 
last  name  being  the  preferable  one.  For  its  use  in  isolating  cells  see  \  245.  « 5 
Micr.  bulletin  and  Sci.  News,  vol.  XII.  |  1895),  pp.  4-5). 

#  309.  Glycerin. — (A).  One  should  procure  pure  glycerin  for  a  mounting  me- 
dium. It  needs  no  preparation,  except  in  some  cases  it  should  be  filtered  through 
filter  paper  or  absorbent  cotton  to  remove  dust,  etc. 

For  preparing  objects  for  final  mounting,  glycerin  50  cc,  water  50  cc,  forms  a 
good  mixture.  For  many  purposes  the  final  mounting  in  glycerin  is  made  in  an 
acid  medium,  viz.,  Glycerin  99  cc,  Glacial  acetic  or  formic  acid,  1  cc. 

By  extreme  care  in  mounting  and  by  occasionally  adding  a  fresh  coat  to  the 
sealing  of  the  cover-glass,  glycerin  preparations  last  a  long  time.  They  are  liable 
to  be  very  disappointing,  however.  In  mounting  in  glycerin  care  should  be  taken 
to  avoid  air-bubbles,  as  they  are  difficult  to  get  rid  of.  A  specimen  need  not  be 
discarded,  however,  unless  the  air-bubbles  are  large  and  numerous. 

Glycerin  Jelly.  —  Soak  25  grams  of  the  best  dry  gelatin  in  cold  water  in  a 
small  agate-ware  dish.      Allow  the  water  to  remain  until  the  gelatin  is  softened 

12 


i78  PREPARATION  OF  REAGENTS.  [CH.   VII. 

usually  takes  about  half  an  hour.  When  the  gelatin  is  softened,  as  may  be  readily 
determined  by  taking  a  little  in  the  fingers,  pour  off  the  superfluous  water  and 
drain  well  to  get  rid  of  all  the  water  that  has  not  been  imbibed  by  the  gelatin. 
Warm  the  softened  gelatin  over  a  water  bath  and  it  will  melt  in  the  water  it  has 
absorbed.  Add  to  the  melted  gelatin  about  5  cc.  of  egg  albumen,  white  of  egg  ; 
stir  it  in  well  and  then  heat  the  gelatin  in  the  water  bath  for  about  half  an  hour. 
Do  not  heat  above  750  or  8j°  C,  for  if  the  gelatin  is  heated  too  hot  it  will  be  trans- 
formed into  meta-gelatin  and  will  not  set  when  cold.  The  heat  will  coagulate  the 
albumen  and  form  a  kind  of  fioculent  precipitate  which  seems  to  gather  all  fine 
particles  of  dust,  etc.,  leaving  the  gelatin  perfectly  clear.  After  the  gelatin  is  clar- 
ified it  should  be  filtered  through  a  hot  flannel  filter  and  mixed  with  an  equal  volume 
of  glycerin  and  5  grams  of  chloral  hydrate  and  shaken  thoroughly.  If  it  is  allowed 
to  remain  in  a  warm  place  [i.  <?.,  in  a  place  where  the  gelatin  remains  melted)  the 
air-bubbles  will  rise  and  disappear. 

In  case  the  glycerin  jelly  remains  fluid  or  semifluid  at  the  ordinary  temperature 
(iS°-20°  C. ),  the  gelatin  has  either  been  transformed  into  meta-gelatin  by  too  high  a 
temperature  or  it  contains  too  much  water.  The  amount  of  water  may  be  lessened 
by  heating  at  a  moderate  temperature  over  a  waterbath  in  an  open  vessel.  This 
is  a  very  excellent  mounting  medium.  Air  bubbles  should  be  avoided  in  mounting 
as  they  do  not  disappear. 

\  jio.  Hematoxylin.— Hematoxylin  is  one  of  the  most  useful  stains  employed  in 
histology.  A  very  excellent  solution  for  ordinary  section  staining  may  be  made  as 
follows  :  Distilled  water  200  cc,  and  potash  alum  7^  grams,  are  boiled  together  for 
5  minutes,  in  an  agate-ware  or  glass  vessel,  and  sufficient  boiled  water  added  to 
bring  the  volume  back  to  200  cc.  After  the  mixture  is  cool,  4  grains  of  chloral  hy- 
drate, and  flTths  gram  of  hematoxylin  crystals,  previously  dissolved  in  20  cc.  of  95% 
alcohol,  are  added.  The  boiling  seems  to  destroy  any  fungi  present  in  the  alum 
or  water,  and  the  chloral  prevents  the  development  of  any  that  may  get  in  after- 
ward, and  this  solution  therefore  is  quite  permanent. 

At  first  the  color  will  be  rather  faint,  but  after  a  week  or  two  it  will  become  a 
deep  purple.  The  deepening  of  the  color  is  more  rapid  if  the  bottle  is  left  uncorked 
in  the  light  and  is  shaken  occasionally. 

If  the  stain  is  too  concentrated  it  may  be  diluted  with  freshly  distilled  water  or 
with  a  mixture  of  water,  alum  and  chloral.  If  the  stain  is  not  sufficiently  concen- 
trated, more  hematoxylin  may  be  added.  With  hematoxylin  of  the  strength  given 
in  the  formula,  sections  are  usually  sufficiently  stained  in  from  one  to  five  minutes. 

As  may  be  inferred  from  what  was  said  above,  the  boiling  is  to  destroy  any  liv- 
ing ferments  present  in  the  water  or  alum,  and  the  chloral  hydrate  is  to  prevent 
the  development  of  germs  which  accidentally  reach  the  solution  after  it  is  made. 

No  precaution  is  necessary  in  using  this  stain  for  sections,  except  that  applicable 
to  all  hematoxylin  solutions,  viz.  :  after  staining,  the  surplus  stain  must  be  very 
thoroughly  washed  away  with  distilled  water;  otherwise  black  granules  or  needles 
will  appear  in  or  upon  the  sections.  If  granules  appear  in  the  preparations  in  spite 
of  the  washing,  it  will  be  well  to  boil  the  solution  three  to  five  minutes  and  filter 
through  paper  or  absorbent  cotton.  The  addition  of  one  or  two  per  cent,  of  chlo- 
ral after  the  boiling  is  also  advantageous.  This  stain  has  not  been  tried  for  dyeing 
in  bulk.  Other  substances  than  chloral  were  tried,  but  not  with  so  good  success. 
(S.  H.  Gage,  Proc.  Amer.  Micr.  Soc,  Vol.  XIV,  1892,  pp.  125-127). 

§311.   Liquid  Gelatin. — Gelatin   or  clear  glue,    75   to  100  grams.     Commercial 


CH.   VII.]  PREPARATION  OF  REAGENTS. 

acetic  acid  (No.  8)  iodcc,  water  ioocc,  95%  alcohol  100  cc,  glycerin  15  to  y  ■ 
Crush  the  glue  and  put  it  into  a  bottle  with  tin-  acid,  and  set  in  a  warm  place,  and 
shake  occasionally.  After  three  or  more  days  add  the  other  ingredients.  This 
lutiou  is  excellent  for  Fastening  paper  to  glass,  wood  or  paper.  The  brush  must  be 
mounted  in  a  quill  or  wooden  handle.  For  labels,  it  is  best  to  use  linen  paper  ol 
moderate  thickness.  This  should  he  coated  with  the  liquid  gelatin  and  allowed  to 
dry.  The  labels  may  be  cut  of  any  desired  size  and  attached  by  Simply  moistening 
them,  as  in  using  postage  stamps. 

Very  excellent  blank  labels  are  now  furnished  by  dealers  in  microscopical  sup- 
plies, so  that  it  is  unnecessary  to  prepare  them  one's  self,  except  for  special  pur- 
poses. 

§312.  Nitric  Acid  Dissociator. — This  is  prepared  by  mixing  80  cc.  of  water  with 
20  cc.  of  strong  nitric  acid.  It  is  used  mostly  in  dissolving  the  connective  tissue  of 
muscle  and  thus  making  it  possible  to  separate  the  fibers.  Alum  water  is  used  as  a 
restrainer  (J  299  and  2.49).    (Gage,  Proc.  Amer.  Micr.  Soc,  Vol.  XI,  (1889),  pp. 

34-45)- 

I  313.  Normal  Salt  Solution  or  Saline  Solution. — Pure  water  from  its  differing 
density  from  the  natural  lymph  acts  injuriously  on  the  tissues.  The  addition  of  a 
little  table  salt,  however,  prevents  this  deleterious  action,  or  greatly  lessens  it, 
hence  the  name  of  normal  salt  solid  ion.  It  is  a  ,,:u%  solution  of  table  salt  (sodium 
chlorid)  in  water  ;  water  1000  cc. ,  salt  6  grams,  or  water  100  cc,  salt  -f6  gram. 

\  314.  Paraffin. — Paraffin  is  of  various  melting  points,  hence  at  the  ordinary  tem- 
perature of  a  laboratory,  that  melting  at  the  lowest  temperature  will  be  moderately 
soft,  hence  soft  paraffin ,  while  that  melting  at  a  higher  temperature  will  be  hard. 
For  the  best  results  one  has  to  mix  hard  and  soft  paraffins.  Usually  a  mixture  of 
9  parts  hard  and  1  part  soft  paraffin  will  give  good  results,  and  may  be  called  im- 
bedding paraffin.  For  chloroform  paraffin,  4  parts  of  imbedding  paraffin  are  mixed 
with  one  part  of  chloroform  (is  301,  272). 

\  315.  Picric-Alcohol. — This  is  an  excellent  hardener  and  fixer  for  almost  all 
tissues  and  organs.  It  is  composed  of  500  cc.  of  water  and  500  cc.  of  95%  alcohol, 
to  which  2  grams  of  picric  acid  have  been  added.  (It  is  a  \%  solution  of  picric 
acid  in  50%  alcohol).  It  acts  quickly,  in  from  one  to  three  days.  (i>  252,  269). 
(Proc.  Amer.  Micr.  Soc,  Vol.  XII  (1890),  pp.  120-122). 

\  316.  Shellac  Cement. — Shellac  cement  for  sealing  preparations  and  for  mak- 
ing shallow  cells  {\  233)  is  prepared  by  adding  scale  or  bleached  shellac  to  95% 
alcohol.  The  bottle  should  be  filled  about  half  full  of  the  solid  shellac  then 
enough  95%  alcohol  added  to  fill  the  bottle  nearly  full.  The  bottle  is  shaken  oc- 
casionally and  then  allowed  to  stand  until  a  clear  stratum  of  liquid  appears  on  the 
top.  This  clear,  supernatant  liquid  is  then  filtered  through  absorbent  cotton,  using 
a  paper  funnel  {\  300,  note),  into  an  open  dish  or  a  wide-mouth  bottle.  To  e\ 
50  cc.  of  this  filtered  shellac,  5  cc.  of  castor  oil  and  5  cc.  of  Venetian  turpentine  are 
added  to  render  the  shellac  less  brittle.  The  filtered  shellac  will  be  too  thin,  and 
must  be  allowed  to  evaporate  till  it  is  of  the  consistency  of  thin  syrup.  It  is  then 
put  into  a  capped  bottle,  and  for  use,  into  a  small  spirit  lamp  (Fig.  13)  .  In  case 
the  cement  gets  too  thick  add  a  small  amount  of  95%  alcohol  or  some  thin  she!' 
The  solution  of  shellac  almost  always  remains  muddy,  and  in  most  cases  it  takes  * 
very  long  time  for  the  fiocculent  substance  to  settle.  One  can  very  quickly  obtain  a 
clear  solution  as  follows  :  When  the  shellac  has  had  time  to  thoroughly  dissolve, 
/.  e. ,  in  a  week  or  two  in  a  warm  place,  or   in   less  time  if  the  bottle  is  frequently 


I  So  MICRO-CHEMISTRY.  {CH.    VII. 

shaken,  a  part  of  the  dissolved  shellac  is  poured  into  a  hottle  and  about  one  fourth 
as  much  gasolin  or  ben/.iu  added  and  the  two  well  shaken.  After  twenty-four 
hours  or  so  the  flocculent,  undissolved  substance  will  separate  from  the  shellac  so- 
lution and  rise  with  the  benzin  to  the  top.  The  clear  solution  may  then  be  siphoned 
off  or  drawn  off  from  the  bottom  if  one  has  an  aspirating  bottle.  (R.  Hitchcock, 
Amer.  Monthly  Micr.  Jour.,  July,  1S84,  p.  131). 

ARRANGING    AND    MOUNTING    MINUTE    OBJECTS. 

',.  317.  Minute  objects  like  diatoms  and  the  scales  of  insects  may  be  arranged  in 
geometrical  figures  or  in  some  fanciful  way,  either  for  ornament  or  more  satisfac- 
tory study.  To  do  this  the  cover-glass  is  placed  over  the  guide.  This  guide  for 
geometrical  figures  may  be  a  net-micrometer  or  a  series  of  concentric  circles.  In 
order  that  the  objects  may  remain  in  place,  however,  they  must  be  fastened  to  the 
cover-glass.  As  an  adhesive  substance,  liquid  gelatin  ($311)  thinned  with  an 
equal  volume  of  50%  acetic  acid  answers  well.  A  very  thin  coating  of  this  is  spread 
on  the  cover  with  a  needle,  or  in  some  other  way,  and  allowed  to  dry.  The  objects 
are  then  placed  on  the  gelatinized  side  of  the  cover  and  carefully  got  into  position 
with  a  mechanical  finger,  made  by  fastening  a  cat's  whisker  in  a  needle  holder. 
For  most  of  these  objects  a  simple  microscope  with  stand  (Figs.  19,  20,  130,  131) 
will  be  found  of  great  advantage.  After  the  objects  are  arranged,  one  breathes 
very  gently  on  the  cover-glass  to  soften  the  gelatin.  It  is  then  allowed  to  dry  and 
if  a  suitable  amount  of  gelatin  has  been  used,  and  it  has  been  properly  moistened, 
the  objects  will  be  found  firmly  anchored.  In  mounting,  one  may  use  Canada  bal- 
sam or  mount  dry  on  a  cell  (§  232,  240).  See  Newcomer,  Amer.  Micr.  Soc.'s  Proc, 
i885,  p.  128;  see  also  E.  H.  Griffith  and  H.  L.  Smith,  Amer.  Jour,  of  Micros.,  iv, 
102,  v,  87;  Amer.  Monthly  Micr.  Jour.,  i,  66,  107,  113.  Cunningham,  The  Micro- 
scope, viii,  1888,  p.  237. 

MICRO-CHEMISTRY    AND    CRYSTALLOGRAPHY — EXPERIMENTS. 

\  318.  The  student  of  science,  and  especially  chemistry,  so  frequently  requires  a 
knowledge  of  the  appearance  of  minute  crystals  to  aid  in  the  determination  of  an 
unknown  substance,  or  for  his  information  in  studying  objects  where  crystals  are 
liable  to  occur,  that  a  few  experiments  have  been  introduced  to  give  him  a  start  in 
preparing  and  permanently  mounting  some  of  the  common  crystals. 

It  is  recommended  that  the  crystals  be  made  in  several  ways,  that  is,  from  alco- 
holic solutions,  aqueous  solutions  saturated  and  dilute,  by  spontaneous  drying  and 
crystallization  and  by  rapid  crystallization  by  the  aid  of  heat.  The  modifications 
in  crystallization  under  these  different  methods  of  treatment  are  frequently  very 
striking. 

In  every  case  the  student  is  advised  to  study  the  appearance  of  the  crystals  in 
the  "mother  liquor."  As  a  rule,  their  characteristics  are  more  clearly  shown  in 
the  "mother  liquor"  than  under  any  other  conditions. 

It  is  of  very  great  advantage  to  examine  all  crystalline  forms  with  polarized  light 
(2  209). 

\  319.  Determination  of  the  Character  of  the  Solid  Sediment  in  Water. —Take 
some  of  the  sediment  from  a  filter  or  allow  a  considerable  volume  of  water  to  stand 


I'll.  /'//.] 


MIL  'RO-CHE.  MIS  TR ) '. 


in  a  tall  glas9  vessel  to  deposit  its  sediment     Take-  a  concentrated  'hop  of  thii 
iment  and  mount  it  on  a  s  1  i « K-  under  a  cover-glass.     Study  the  preparation  with  the 
microscope.     Probably  there  will  be  an  abundance  of  animal  and  vegetable  1 

well  as  of  solid  sediment.      Put  a  drop  of  dilute  sulphuric  a   id      .  /,  idurtt  su/phtiri- 

cum,  i.e.,  strong  sulphuric  arid  i  gram,  water  9  grams)  at  the  edge  of  the  1 


Fig.  139.  Czapski's  Ocular  Ms-diaphragm  with  cross  h 
for  e  1  amining  and  act  urately  determining  the  axial  im 
small  crystals.       The  iris  diaphragm   enables   the  1  r  to 

wake  the  field  as  large  or  small  as  desired. 

A.  Longitudinal  section. 

B.  Transection,  showing  the  cross  lines  ant!  the  iris  dia- 
phragm with  the  projecting  part  at  the  left,  by  'which  the  dia- 
phragm is  opened  and  closed.     {Zeiss'  Catalog,  Xo.  30). 


and  at  the  opposite  edge  a  small  piece  of  the  lens  paper  (Fig.  128).  The  acid  will 
gradually  diffuse,  and  if  the  solid  particles  are  carbonate  of  lime,  minute  bubbles 
will  be  seen  to  be  given  off.  If  they  are  silica  or  clay  no  change  will  result.  Sul- 
phuric acid  is  recommended  for  this,  as  the  microscope  would  be  far  less  liable  to 
injury  than  as  if  some  acid  gi%'ing  off  fumes  were  used. 

§320.  Herapath's  Method  of  Determining  Minute  Quantities  of  Quinine. —  For 
a  so-called  test  fluid  12  cc.  of  glacial  acetic  acid,  4  cc.  of  95%  alcohol,  and  7  drops 
of  dilute  sulphuric  acid  (§  319)  are  mixed.  A  drop  of  the  test  fluid  is  put  on  a  slide 
and  a  very  minute  amount  of  quinine  added.  After  this  is  dissolved,  add  an  ex- 
tremely minute  drop  of  an  alcoholic  solution  of  iodine.  "The  first  effect  is  the 
production  of  the  yellow  cinnamon-colored  compound  of  iodine  and  quinine,  which 
forms  as  a  small  circular  spot  ;  the  alcohol  separates  in  little  drops,  which,  by  a 
sort  of  repulsive  movement,  drive  the  fluid  away  ;  after  a  time  the  acid  liquid  again 
flows  over  the  spot,  and  the  polarizing  crystals  of  sulphate  of  iodo-quinine  are  slow  - 
ly  produced  in  beautiful  rosettes.  This  succeeds  best  without  the  application  of 
heat."  Dr.  Herapath  used  this  method  to  determine  the  presence  of  quinine  in 
the  urine  of  patients  under  quinine  treatment.  See  Hogg,  p.  150;  Quarterly  Jour. 
Micr.  Sc,  vol.  ii,  pp.  13-18.  For  further  papers  on  micro-chemistry  by  Dr.  H< 
path,  see  the  Royal  .Society's  Catalog  of  Scientific  Papers. 

\  321 .    List  of  Substances  for  the  Study  of  Crystallography  with  the  Microscope. 
The  substances  are  crystalized  on  the  cover-glass  in  all  cases,  and  in  all  cases,  1 
cept  where  otherwise  stated,  a  saturated  aqueous  solution  of  the  substances  was 
first  prepared. 

1.  Ammonium  chlorid  ;    2.  Ammonium    copper  chlorid  ;    3.   barium    chlorid  . 
4.  Cobalt  chlorid  (beautiful  crystals  obtained  by  mixing  the  saturated  aqueous 
lution  with  an  equal  volume  of  95%  alcohol).     Crystallization  in  a  current  of  dry 


M  >-t  of  the  chemicals  here  name  1  weir  suggested  to  the  writer  by  Prof    I.    M. 
Dennis,  of  the  Chemical  Department. 


182  MICRO-CHEMISTRY.  \CH.    VII. 

air  some  distance  above  an  alcohol  or  Runsen  flame  ;  mount  in  xylene  balsam 
[\  300),  or  one  ma}'  fuse  the  salt  with  the  balsam  (§  300);  5.  Copper  acetate; 
Mount  dry  {\  231);  6.  Copper  sulphate.  Crystals  much  more  satisfactory  when 
examined  in  the  "mother  liquor."  7.  Lead  nitrate;  8.  Mercuric  chlorid  (corro- 
sive sublimate1,  mount  in  xylene  balsam  {\  300,  240).  9.  Nickel  nitrate,  obtain 
crystals  by  heating;  mount  in  xylene  balsam  ({j  240,  300)  ;  10.  Potash  alum;  ir. 
Potassium  chlorate;  12.  Potassium  dichromate.  Compare  specimen  crystallized 
by  heat  and  spontaneously;  mount  dry  or  in  xylene  balsam  {\  300).  13.  Po- 
tassium iodide.  Dilute  with  one  or  two  volumes  of  water,  and  crystallize  by  heat. 
14.  Potassium  nitrate;  15.  Potassium  oxalate;  16.  Potassium  sulphate;  17.  Sali- 
cin.  Fuse  the  dry  salicin  on  the  cover-glass,  mount  dry,  or  preferably  fuse  in 
balsam  (§  300).  18.  Salicylic  acid.  Make  a  10%  solution  in  95%  alcohol  ;  let  it 
crystallize  spontaneously  in  the  air;  mount  dry  (J  231).  19.  Sodium  chlorid 
(common  salt).  Mix  sat.  aq.  sol.  with  one  or  two  volumes  of  water,  and  heat  ; 
mount  dry  or  in  balsam.     20.   Sulphonal  ('i  21S). 

\  322.  For  directions  and  hints  in  micro-chemical  work  and  crystallography, 
consult  the  various  volumes  of  the  Journal  of  the  Roy.  Micr.  Soc.  ;  Zeitschrift  fiir 
physiologische  Chemie,  and  other  chemical  journals  ;  Wormly  ;  Klement  &  Re- 
nard ;  Carpenter-Dallinger  ;  Hogg;  Behrens,  Kossel  und  Schiefferdecker  ;  Frey  ; 
Dana,  and  other  works  on  mineralogy  ;  Davis. 


CHAPTER  VIII. 


PHOTOMICROGRAPHY    AND    PHOTOGRAPHY    WITH    A 

VERTICAL  CAMERA.* 


APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER. 

Compound  microscope  with  achromatic  condenser;  Achromatic  and  apocbro- 
niaticobjec  ives  ;  Oculars,  ordinary  and  projection  ;  Lamp  and  bull's-eye  condeusei 
of  some  form  ;  Photo  nricrographic  camera,  and  an  ordinary  copying  camera  ;  Fo 
cusing  glass;  dry  plates,  developer,  fixer,  trays,  dark  room,  and  the  other  things 
needed  for  photography,  like  printing  frames,  etc.,  etc.  ;  Photographic- objectives, 
one  for  large  objects  and  one  for  small  objects  to  be  magnified  from  two  to  fifteen 
diameters. 

\  323.  Nothing  would  seem  more  natural  than  that  the  camera,  armed  with  a 
photographic  objective  or  with  a  microscopic  objective,  should  be  called  into  the 
service  of  science  to  delineate  with  all  their  complexity  of  detail,  the  myriads  of 
forms  studied.  Indeed,  the  very  first  pictures  made  on  white  paper  and  white 
leather,  sensitized  by  silver  nitrate,  were  made  by  the  aid  of  a  solar  microscope 
(1S02).  The  pictures  were  made  by  Wedgwood  and  Davy,  and  Davy  says  :  "  I  have 
found  that  images  of  small  objects  produced  by  means  of  the  solar  microscope  may 
be  copied  without  difficulty  on  prepared  paper."  f 

*  Considerable  confusion  exists  as  to  the  proper  nomenclature  of  photography 
with  the  microscope.  In  Germany  and  France  the  term  micro- photography  is  very 
common,  while  in  English  photo-micrography  and  micro-photography  mean  difer- 
ent  things.  Thus  :  A  photo-micrograph  is  a  photograph  of  a  small  or  microscopic 
object,  usually  made  with  a  microscope  and  of  sufficient  si/.a  for  observation  with 
the  unaided  eye;  while  a  micro-photograph  is  a  small  or  microscopic  photograph 
of  an  object,  usually  a  large  object,  like  a  man  or  woman,  and  is  designed  to  lx- 
looked  at  with  a  microscope. 

Dr.  A.  C.  Mercer,  in  an  article  in  the  Proc.  Amer.  Micr.  Soc.,  18S6,  p.  [31,  says 
that  Mr.  George  Shadbolt  made  this  distinction.  See  the  Liverpool  and  Manches- 
ter Photographic  Journal  now  British  Journal  of  Photography  ,  Aug.  is.  1858,  p. 
203;  also  Sutton's  Photographic  Notes,  Vol.  Ill,  1858,  pp.  205-20N.  On  p.  208  of 
the  last,  Shadbolt's  word  '•Photomicrography"  appears.  Dr.  Mercer  puts  the 
case  very  neatly  as  follows  :  "  A  photo  micrograph  is  a  macroscopic  photograph  of 
a  microscopic  object ;  a  micro-photograph  is  a  microscopic  photograph  of  a  ma 
scopic  object."    See  also  Medical  News,  Jan.  27,  [894,  p.  108. 

1  In  a  most  interesting  paper  by  A.  C.  Mercer  on  "  The  Indebtedness  of  Phi 
raphy  to  Microscopy,   Photographic  Times  Almanac,  [887,  it  is  shown  that :     'To 


[84  PHOTO-MICROGRAPHY.  \_CH.   VIII. 

Thus  among  the  very  first  of  the  experiments  in  photography  the  microscope 
was  called  into  requisition.  And  naturally,  plants  and  motionless  objects  were 
photographed  in  the  beginnings  of  photography  when  the  time  of  exposure  re- 
quired was  very  great. 

At  the  present  time  photography  is  used  to  an  almost  inconceivable  degree  in  all 
the  arts  and  sciences  and  in  pure  art.  Even  astronomy  finds  it  of  the  greatest 
assistance. 

Although  first  in  the  field,  Photo-Micrography  has  been  least  successful  of  the 
branches  of  photography.  This  is  due  to  several  causes.  In  the  first  place,  mi- 
croscopic objectives  have  been  naturally  constructed  to  give  the  clearest  image  to 
the  eye,  that  is  the  visual  image  as  it  is  sometimes  called,  is  for  microscopic  obser- 
vation, of  prime  importance.  The  actinic  or  photographic  image,  on  the  other 
hand,  is  of  prime  importance  for  photography.  Then  for  the  majority  of  micro- 
scopic objects  transmitted  light  {\  50)  must  be  used,  not  reflected  light  as  in  ordin- 
ary vision.  Finally,  from  the  shortuess  of  focus  and  the  small ness  of  the  lenses, 
the  proper  illumination  of  the  object  is  accomplished  with  some  difficulty,  and  the 
fact  of  the  lack  of  sharpness  over  the  whole  field  with  any  but  the  lower  powers, 
have  all  combined  to  make  photo-micrography  less  successful  than  ordinary 
macro-photography.  So  tireless,  however,  have  been  the  efforts  of  those  who  be- 
lieved in  the  ultimate  success  of  photo-micrography,  that  now  the  ordinary  achro- 
matic objectives  and  ortho-chromatic  or  is  achromatic  plates  give  very  good  results, 
while  the  apochromatic  objectives  with  projection  oculars  give  excellent  results, 
even  in  hands  not  especially  skilled.  The  problem  of  illumination  has  also 
been  solved  by  the  construction  of  achromatic  and  apochromatic  condensers  and 
by  the  electric  and  other  powerful  lights  now  available.  There  still  remains  the 
the  difficulty  of  transmitted  light  and  of  so  preparing  the  object  that  structural  de- 
tails shall  stand  out  with  sufficient  clearness  to  make  a  picture  which  shall 
approach  in  definitetiess  the  drawing  of  a  skilled  artist. 

The  writer  would  advise  all  who  wish  to  undertake  photo-micrography  seriously, 
to  study  samples  of  the  best  work  that  has  been  produced.  Among  those  who 
showed  the  possibilities  of  photo-micrographs  was  Col.  Woodward  of  the  U.  S. 
Army  Medical  Museum.  The  photo-micrographs  made  by  him  and  exhibited  at 
the  Centennial  Celebration  at  Philadelphia  in  1876,  serve  still  as  models,  and  no 
one  could  do  better  than  to  study  them  and  try  to  equal  them  in  clearness  and 
general  excellence.  According  to  the  writer's  observation  no  photo-micrographs 
of  histological  objects  have  ever  exceeded  those  made  by  Woodward,  and  most  of 
them  are  vastly  inferior.  It  is  gratifying  to  state,  however,  that  at  the  present 
time  (  1896)  many  original  papers  are  partly  or  wholly  illustrated  by  photo-micro- 
graphs, and  no  country  has  produced  works  with  photo-micrographic  illustrations 
superior  to  those  in  "Wilson's  Atlas  of  Fertilization  and  Karyokinesis "  and 
"Starr's  Atlas  of  Nerve  Cells,"  issued  by  the  Columbia  University  Press. 


briefly  recapitulate,  photograph}-  is  apparently  somewhat  indebted  to  microscopy 
for  the  first  fleeting  pictures  of  Wedgwood  and  Davy  [1S02],  the  first  methods  of 
producing  permanent  paper  prints  [Reede,  1837-1S39],  the  first  offering  of  prints 
for  sale,  the  first  plates  engraved  after  photographs  for  the  purpose  of  book  illustra- 
tion [Donne  &  Foucault,  1S45],  the  photographic  use  of  collodion  [Archer  &  Dia- 
mond, 185 1],  and  finally,  wholly  indebted  for  the  origin  of  the  gelatino-bromide 
process,  greatest  achievement  of  them  all  [Dr.  R.  L.  Maddox,  1871].  See  further 
for  the  history  of  Photo-micrography,  Neuhauss,  also  Bousfield. 


CH.   ] '///.]  PHOTO  Mh  Ri  n;h>.  !/•// ) . 

Is  the  difficulties  of  photo-inicrography  are  so  much  greater  than   of  ordinary 
photography,  the  advice  is  almost  univei  >al  that  no  one  should  try  to  learn  phol 
graphy  and  photo-micrography  at  the  same  time,  but  that  one  should   learn  the 
processes  of  photography  by  making  portraits,  landscapes,  copying  drawings 
and  then  when  the  principles  are  learned  one  can   undertake  the  more  difficult 
problem  of  photo-micrography  with  some  hope  "I  success. 

The  advice  of  Sternberg  is  so  pertinent  and  judicious  that  it  is  reproduced 
"Those  who  have  had  no  experience  in  making  photo-micrographs  are  apt  t<»  • 
pect  too  much  and  to  underestimate  the  technical  difficulties.  Objects  which 
under  the  microscope  give  a  beautiful  picture,  which  we  desin  to  reproduce  by 
photography,  may  be  entirely  unsuited  for  the  purpose.  In  photographing  with 
high  powers  it  is  necessary  that  the  objects  to  be  photographed  be  in  a  single  plane 
ami  not  crowded  together  and  overlying  each,  other.  For  this  reason  photograph- 
ing bacteria  in  sections  presents  special  difficulties  and  satisfactory  results  can  only 
be  obtained  when  the  sections  are  extremely  thin  and  the  bacteria  well  stained. 
Even  with  the  best  preparations  of  this  kind  much  care  must  be  taken  in  selecting 
a  field  for  photography.  It  must  be  remembered  that  the  expert  microscopist,  in 
examining  a  section  with  high  powers,  has  his  finger  on  the  fine  adjustment  screw 
and  focuses  up  and  down  to  bring  different  planes  into  view.  lie  is  in  the  habit  ol 
fixing  his  attention  on  the  part  of  the  field  which  is  in  focus  and  discarding  the 
rest.  Hut  in  a  photograph  the  part  of  the  field  not  in  focus  appears  in  a  promi- 
nent way  which  mars  the  beaut}-  of  the  picture." 

APPARATUS    FOR    PHOTOMICROGRAPHY. 

\  324.  Camera. — For  the  best  results  with  the  least  expenditure  of  time  one  of 
the  cameras  especially  designed  for  photo-micrography  is  desirable  but  is  not  by 
any  means  necessary  for  doing  good  work.  An  ordinary  photographic  camera, 
especially  the  kind  known  as  a  copying  camera,  will  enable  one  to  get  good  results, 
but  the  trouble  is  increased,  and  the  difficulties  are  so  great  at  best,  that  one  would 
do  well  to  avoid  as  many  as  possible  and  have  as  good  an  outfit  as  can  be  afforded. 

The  first  thing  to  do  is  to  test  the  camera  for  the  coincidence  of  the  plane  occu- 
pied by  the  sensitive  plate  and  the  ground  glass  or  focusing  screen.  Cameras  even 
from  the  best  makers  are  not  always  correctly  adjusted.  By  using  a  straight  edge 
of  some  kind,  one  can  measure  the  distance  from  the  inside  or  ground  side  of  the 
focusing  screen  to  the  surface  of  the  frame.  This  should  be  done  all  around  tosee 
if  the  focusing  screen  is  equally  distant  at  all  points  from  the  surface  of  the 
frame.  If  it  is  not  it  should  be  made  so.  When  the  focusing  screen  has  been  • 
amined,  an  old  plate,  but  one  that  is  perfectly  flat,  should  be  put  into  the  plate 
holder  and  the  slide  pulled  out  and  the  distance  from  the  surface  of  the  plate 
holder  determined  exactly  as  for  the  focusing  screen.  If  the  distance  is  not  the 
same,  the  position  of  the  focusing  screen  must  be  changed  to  correspond  with  that 
of  the  glass  in  the  plate  holder,  for  unless  the  sensitive  surface  occupies  e\  ictly 
the  position  of  the  focusing  screen  the  picture  will  not  be  sharp  no  matter  how 
accurately  one  may  focus  Indeed,  so  necessary  is  the  coincidence  ol  the  plane  of 
the  focusing  screen  and  sensitive  surface  that  some  photo-micrographers  put  the 
focusing  screen  in  the  plate  holder.  focus  the  image  ami  then  put  the  BetlSlUve 
plate  in  the  holder  and  make  the  exposure     Cox  1.     This  would  b<  ■'  tni" 

the  older  forms  of  plate  holders,  but  not  with  the  double  plate  holders  mostly  used 
at  the  present  day. 


1 86 


PI  10  TO-MICROOHAPA  V. 


\_CH.   VIII. 


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Fig.  143. 


CI  I.   \  'III.  ]  /'//< )  T<  >  MICROGRAPH  i '. 

Fig,  i  i.v   Walmsley's  Improved  Photo- Micrographic  Camera.    In  this  jigm 
shown  an  excellent  form  of  photo  micrographic  camera  for  use  with  a  horizontal 
microscope.     It  has  the  advantage  of  the  possibility  of  a  very  long   bellows  or  a 
shorter  one  as  the  need  of  the  specal  case  demands.     It  is  arranged  fot  photo-mi 
crographic  work  with  the  microscope  or  a  wide  angled  objective  or  fot  copying  and 
slightly  enlarging  or  diminishing  t  drawing,  etc.,  with  an  ordinary  photographic 
objective.     It  is  a/so  arranged  for  making  lantern  slides.     .  I  very  simple  arrange- 
ment has  hern  adopted  for  focusing  when  the  bellows  is  pulled  out  so  far  that  one 
cannot  reach  the  fine  adjustment,  and  it  works  with  great  smoothness.     ".  I 
is  turned  in  the  periphery  of  the  fine  adjustment  screw,  around  which  a  small , 
is  passed,  and  carried  through  a  succession  of  screw  eyes  on  either  side  of  the  base- 
board  to  the  rear,  where  a  couple  of  small  leaden  weights  arc  attached  to  its  ends, 
thus  keeping  the  cord  taut.     A   -eery  slight  pull  on  either  side,  -whilst  th 
fixed  upon  the  image  on  the  screen,  suffices  to  adjust  the  focus  with  the  utmost 
exactness.     If  preferred,  a  rod  running  the  entire  length  of  the  camera-bed,  and 
terminating  at  the  rear  end  with  a  milled  head,  whilst  on  the  other  is  a  grooved 
pulley,  carrying    a  cord  or  belt  zvhich  also  passes  around  the  groove  in  the  milled 
head  of  the  fine  adjustment,  may  be  substituted  for  the  cord  and  weights.     This 
arrangement  [shown  in  the  figure]  is  more  costly,  and  probably  no  better  in  actual 
service  than  that  with  the  cord  and.  rceighls.'"     (From  Mr.  Walmsley.  i 

$325.  Work  Room. — It  is  almost  self-evident  that  the  camera  must  he  in  some 
place  free  from  vibration.  Frequently  a  basement  room  where  the  camera  table 
may  rest  directly  on  the  ground  or  on  a  pier  is  an  excellent  situation.  Such  a  sit- 
uation is  almost  necessary  for  the  best  work  with  high  powers.  For  those  living 
in  cities,  a  time  must  also  be  chosen  when  there  are  no  heavy  vehicles  moving  in 
the  streets.  For  less  difficult  work  an  ordinary  room  in  a  quiet  part  of  the  house 
or  laboratory  building  will  suffice. 

\  326.  Arrangement  and  Position  of  the  Camera  and  the  Microscope. — F'or  the 
greater  number  of  photo-micrographs,  a  horizontal  camera  and  microscope  are  to 
be  preferred  as  one  may  then  vary  the  length  of  the  bellows  at  will  and  still  pre- 
serve steadiness  (Fig.  143).  F'or  some  specimens,  however,  it  is  necessary  to  keep 
th*  microscope  in  a  verticil  position,  hence  to  phonograph,  the  camera  must  also 
b^  vertical.  Very  excellent  arrangements  were  perfected  long  ago,  especially  by 
the  French.     (.See  Moitessier). 

Vertical  photo-micrographic  cameras  are  now  very  commonly  made,  and  by  some 
firms  only  vertical  cameras  are  produced.  They  are  exceedingly  convenient,  and 
do  not  require  so  great  a  disarrangement  of  the  microscope  to  make  the  picture. 
Van  Ileurck  advises  their  use,  then  whenever  a  structure  is  shown  with  especial 
excellence  it  is  photographed  immediately.  The  variation  in  size  of  the  picture  is 
obtained  by  the  objective  and  the  projection  ocular  rather  than  by  length  of  bel- 
lows (see  below).  |  Fig.  141).  It  must  not  be  forgotten,  however,  that  penetration 
varies  inversely  as  the  square  of  the  power,  and  only  inversely  as  the  numerical 
aperture  {'{  29),  consequently  there  is  a  real  advantage  in  using  a  low  power  of 
great  aperture  and  a  long  bellows  rather  than  an  objective  of  higher  power  with  a 
shorter  bellows.     (See  Carpenter-Dallinger,  pp.  318-319). 

#327.  Microscope. —For  convenience  and  rapidity  of  work  a  microscope  with 
mechanical  stage  is  very  desirable.  It  is  also  an  advantage  to  have  a  tube  of  large 
diameter  so  that  the  field  will  not  be  too  greatly   restricte'l.      In  some  microsco] 


[88 


/  V/0  TO-MICROGRA  PH  Y. 


ICH.   VIII. 


the  tube  is  removable  almost  to  the  nose-piece  to  avoid  interfering  with  the  size  of 
the  image.  The  substage  condenser  should  be  movable  on  a  rack  and  pinion. 
The  microscope  should  have  a  flexible  pillar  for  work  in  a  horizontal  position. 
While  it  is  desirable  in  all  cases  to  have  the  best  and  most  convenient  apparatus 
that  is  made,  it  is  not  by  any  means  necessary  for  the  production  of  excellent 
work.     A  simple  stand   with   flexible  pillar  and  good  fine  adjustment  will  answer. 


Fig.   144. 

Fig.  144.  Leitz*  Photo  -  Micrographic  Camera  for  Vertical  Microscope  {from 
Leitz'  American  House,  New  York).  The  camera  may  be  raised  or  lowered  to 
adapt  it  to  various  stands.  The  microscope,  as  shown,  rests  on  the  base  of  the  cam- 
era support,  thus  eliding  steadiness,  and  the  simultaneous  vibration  of  the  entire 
apparatus,  if  any  should  occur. 

With  this  instrument  the  focusing  can  be  done  with  the  hand  on  the  fine  adjust- 
ment, as  in  ordinary  microscopical  study. 


CH.    I'll  I.  | 


/•IIOTO-MICh'OdA-.U'llV. 


\  328.  Objectives  and  Oculars  for  Photo-Micrography. — The  belief  is  almost 
universal  that  the  apochromatic  objectives  art-  im>-.t  satisfactory  for  photography. 
They  are  employed  fortius  purpose  with  a  special  projection  ocular.  Two  very 
low  powers,  one  of  ,;s  ami  one  of  7"  millimeters  equivalent  focus  are  used  without 
any  ocular  i  Fig.  149).  Some  of  the  best  work  thai  has  ever  been  done,  hour 
was  done  with  achromatic  objectives  (work  of  Woodward  ami  others  .  One  n< 
not  desist  from  undertaking  photo  micrography  if  he  has  good  achromatic  ob- 
jectives. From  a  somewhat  extended  series  of  experiments  with  the  objectives oi 
many  makers  the  good  modern  achromatic  objectives  were  found  to  give  excellent 
results  when  used  without  an  ocular.  Most  of  them  also  gave  good  results  with 
projection  oculars,  although  it  must  he  said  that  the  best  results  were  obtained  w  ith 
the  apochromatic  objectives  and  projection  oculars.  It  does  not  seem  to  require 
so  much  skill  to  get  good  results  with  the  apochromatics  as  with  the  achromatic 
objectives.  The  majority  of  photo-micrographers  do  not  use  the  Huygenian  ocu- 
lars in  photography,  although  excellent  results  have  been  obtained  with  them.  An 
amplifier  is  sometimes  used  in  place  of  an  ocular.  Considerable  experience  is 
necessary  in  getting  the  proper  mutual  position  of  objective  and  amplifier.  The 
introduction  of  oculars  especially  designed  for  projection,  has  led  to  the  discarding 
of  ordinary  oculars  and  of  amplifiers.  However  the  projection  oculars  of  Z< 
restrict  the  field  very  greatly,  hence  the  necessity  of  using  the  objective  alone  for 
large  specimens/- 

Fig.  145.  Projection  Oculars  with  section  re- 
moved to  show  the  construction.  Below  arc 
shown  the  upper  end  with  graduated  circle  to 
indicate  the  amount  of  rotation  found  necessary 
to  focus  the  diaphragm  on  the  screen.  A'o.  .?, 
Xo.  /.  The  numbers  indicate  the  amount  the 
ocular  magnifies  the  image  formed  by  the  ob- 
jective as  with  the  compensation  oculars. 
{Zeiss'  Catalog,  No.  30). 

\  329.  Difference  of  Visual  and  Actinic  Foci. 
Formerly  there  was  much  difficulty  exper- 
ienced in  photo-micrographing  on  account  of 
the  difference  in  actinic  and  visual  foci.  Mod- 
ern objectives  are  less  faulty  in  this  respect 
and  the  apochromatics  are  practically  free 
from  it.  Since  the  introduction  of  orthochromatic  or  isochromatic  pi 
and,    in    many    cases     the     use     of    colored     screens,     but     little    trouble    has 


No.  2 


*The  comparative  study  bo'.h  with  projection  oculars,  .and  without  an  ocular  were 
made  with  the  achromatic  objectives  25  mm.  (  1  inch  1,  is  mm.  1  ;4  inch),  5  nun.  1 
to  \ inch)  and  2 mm.  ,'_,  (inch)  homogeneous  immersion  of  the  Bauscb  &  Lomb 
Optical  Co.;  Gundlach  Optical  Co.;  Leitz  ;  Reichert  ;  Winkel  and  Zeiss.  Good 
results  were  obtained  with  all  of  these  objectives  both  with  and  without  projection 
oculars.  The  objectives  of  Spencer  ami  Smith,  especially  constructed  lor  photo- 
micrography, are  highly  spoken  of  by  Piffard  (Jour.  Roy.  Micr.  Soc,  [892  is>. 
The  writer  has  not  hail  the  opportunity  of  comparing  these  with  those  mentii  ned. 
Piffard  spoke  highly  of  the  work  of  Wales  also.  From  the  known  excellence  ol 
the  work  of  these  opticians  one  would  expect  good  results. 


190 


PHOTO  MICROGRAPHY. 


\_CH.    VIII. 


arisen  from  <li (Terences  in  the  foci.  This  is  especially  true  when  mono-chro- 
matic light  and  even  when  petroleum  light  is  used.  In  case  the  two  foci  are  so 
unlike  in  an  objective,  it  would  be  better  to  discard  it  for  photography  altogether, 
for  the  estimation  of  the  proper  position  of  the  sensitive  plate  after  focusing  is 
only  guess  work  and  the  result  is  only  mere  chance.  If  sharp  pictures  cannot  be 
obtained  with  an  objective  when  lamp  light  and  orthochromatic  plates  are  used 
the  fault  may  not  rest  with  the  objective  but   with   the  plate  holder  and  focusing 


Fig.  146.      IValmsley's  Autograph  Photo-Micrograph ic  Camera  in  a  vertical  po- 
sition.     Compare  Fig.  1  jj   (From  the  /'roc.  Amer.  Micr.  Soc.   Vol.  XVII,  1S95). 


CH   17//.}  PHOTO-MICROGRAPHY.  191 

screen.     They  should  be  carefully  tested   10  see  if  there  is  coincidence  in  position 
of  tlie  focusing  screen  and  the  sensitive  film  as  described  in    \  524. 

jo.  Apparatus  for  Lighting,— For  1<>\\  power  work  (35  mm.  ami  longer 
focus)  ami  for  large  objects  some  form  of  bull's  eye  condenser  is  desirable  although 
fairly  good  work  may  be  done  with  diffused  1  i j^l  1 1  or  lamp  li.^ht  reflected  by  a  mir- 
ror. If  a  hull's  eye  is  used  it  should  he  as  nearly  achromatic  as  possible.  The 
engraving  glass  shown  in  Fig.  [55  answers  very  well  for  large  objects.  For  smaller 
ohjects  a  Steinheil  lens  combination  gives  a  more  brilliant  light  and  one  also  m<  • 
nearly  achromatic.  For  bigh  power  work  all  are  agreed  that  nothing  will  take  tin- 
place  of  an  achromatic  condenser.  This  may  be  simply  an  achr  matic  COliden 
but  preferably  it  should  he  an  apochromatic  condenser.  Whatever  the  form  of  the 
condenser  it  should  possess  diaphragms  so  that  the  aperture  of  the  condenser  may 
he  varied  depending  upon  the  aperture  of  the  objective.  For  a  long  time,  objec- 
tives have  been  used  as  achromatic  condensers,  and  they  are  very  satisfactory, 
although  less  convenient  than  a  special  condenser  whose  aperture  is  great  enough 
for  the  highest  powers  and  capable  of  being  reduced  by  means  of  diaphragms  to 
the  capacity  of  the  lower  objectives.  It  should  also  be  capable  of  accurate  center- 
ing. 

\  331.  Light  Filters  or  Color  Screens.— These  are  solutions  or  suitably  stained 
collodion  or  gelatin  films  placed  between  the  source  of  illuiniiiatii.11  and  the  ob- 
ject. It  does  not  make  much  difference  where  the  color  screen  is  placed  provided 
no  light  reaches  the  object  which  has  not  passed  through  the  filter.  The  purpose 
of  the  color  screen  or  filter  is  to  take  out  the  excessive  number  of  blue  and  violet 
rays  so  that  the  more  slowly  acting  red,  yellow  and  green  may  have  time  to  pro- 
duce the  appropriate  chemical  changes  in  the  sensitive  plate.  This  action  of  the 
longer  waves  (see  under  spectroscope  \  179-19S),  is  greatly  aided  by  the  isochro- 
matic  or  orthoehromatic  plates  which  are  especially  treated  so  that  they  will  be 
sensitive  to  the  longer  waves  as  well  as  to  the  shorter,  blue  and  violet  waves.  This 
is  why  it  is  so  necessary  in  manipulating  the  plates  to  avoid  exposing  them  to 
any  light  whatsoever  in  putting  them  into  the  plate  holder,  developing,  etc. 

The  color  screen  used  by  Dr.  Learning  in  preparing  the  negatives  for  the  plates 
in  Wilson's  and  Starr's  atlases  was  made  by  staining  a  lantern  slide  plate  from 
which  all  the  silver  salts  had  been  removed,  with  an  alcoholic  solution  of  tropaeo- 
Hit  and  then  after  drying,  Canada  balsam  and  a  coverglass  were  applied.  <  Hhers 
have  recommended  collodion  stained  with  aurantia.  The  purpose  of  these  is  to 
filter  out  the  greater  number  of  the  blue  and  violet  rays  and  then  increase  the  lime 
of  exposure  from  2  to  5  times,  depending  on  the  thickness  of  the  color  screen. 
Color  screens  and  color  cells  are  furnished  by  various  makers.  None  of  tlu-m 
answers  for  all  preparations  and  for  some  preparations  they  are  unnecessary. 

\  332.  Objects  Suitable  for  Photo-micrographs  —While  almost  any  large  object 
may  be  photographed  well  with  the  ordinary  camera  and  photographic  objective, 
only  a  small  part  of  the  objects  mounted  for  microscopic  study  can  be  pboto-mi- 
crographed  satisfactorily.  Many  objects  that  give  beautiful  and  satisfactory  imagi  a 
when  looking  into  the  microscope  and  constantly  focusing  with  the  tine  adjust- 
ment, appear  almost  without  detail  on  the  screen  of  the  photo  micrograph ic  cam- 
era and  in  the  photo-micrograph. 

If  one  examines  a  series  of  photo-micrographs  the  chances  are   that    the  gn 
number  will  be  of  diatoms,  plant  sections  or  preparations  of  inserts       That  is,  t'. 
are  of  objects  having  sharp  details  and  definite  outlines,  so  th  it  contrast  and  d< 


[92 


/  V/0  TO-  M/CROC  RAPH  ) r. 


[CH.    VIII. 


niteness  may  tie  readily  obtained.  .Stained  microbes  also  furnish  favorable  objects 
when  mounted  as  cover  »lass  preparations. 

Preparations  in  animal  histology  must  approximate  as  nearly  as  possible  to  the 
conditions  more  easily  obtained  with  vegetable  preparations.  That  is,  they  must 
be  made  so  thin  and  be  so  prepared  that  the  cell  outlines  will  have  something  of 
the  dafiuiteness  of  vegetable  tissue.  It  is  useless  to  expect  to  get  a  clear  photo- 
graph of  a  section  in  which  the  details  are  seen  with  difficulty  when  studying  it 
under  the  microscope  in  the  ordinary  way. 

Many  sections  which  are  unsatisfactory  as  wholes,  ma}'  nevertheless  have  parts 
in  which  the  structural  details  show  with  satisfactory  clearness.  In  such  a  case  the 
part  of  the  section  showing  details  satisfactorily  should  be  surrounded  by  a  delicate 
ring  by  means  of  a  marker  (see  Figs.  6t-66).  If  one's  preparations  have  been 
carefully  studied  and  the  special  points  in  them  thus  indicated,  they  will  be  found 
far  more  valuable  both  for  ordiu  iry  demonstration  and  for  photography.  The 
amount  of  time  saved  by  marking  one's  specimens  can  hardly  be  overestimated. 
The  most  satisfactory  material  for  making  the  rings  is  shellac  colored  with  an  alco- 
holic solution  of  one  of  the  anilins,  blue  or  green,  then  in  studying  the  prepara- 
tion one  can  see  it  even  where  covered  by  the  ring. 


Fig.  147.  Walmsleys  Autograph  Photo  micrographic  Camera  in  a  horizontal 
position.  A  microscope  la>np  and  bull's-eye  condenser  are  in  position.  Compare 
Fig.  148  in  \  I 'roc.  Amcr.  Micr.  Soc,   Vol.  XVII,  /S95). 

\  333.  Light. — The  strongest  available  light  is  sunlight.  That  has  the  defect  of 
not  always  being  available,  and  of  differing  greatly  in  intensity  from  hour  to  hour, 
day  to  day  and  season  to  season.     The  sun  does  not  shine  in  the  evening  when 


ch.  iv/r.] 


/■//< >/•<>  .t//rA'(x,A'.  //'//>• 


many  workers  find  the  only  opportunity  for  work.  Following  the  BUnlight,  the 
electric  li^ht  is  tin-  most  intense  of  the  available  limits.  Then  comes  magm 
inn,  the  lime  li^'it,  the  j;;is-j^1o\v  or  Well6bacfa  light,  and  lastly,  petroleum 
light.  The  last  is  excellent  for  the  majority  of  low  and  moderate  power  work. 
And  even  for  2  mm.  homogeneous  immersion  objectives,  the  time  of  exposure 
not  excessive  for  many  specimens  ( \]/2  to  3  minutes).  This  light  is  also  cheapest 
and  most  available. 

Fig.     i  is.     Vertical     Photo- Micrographu 

Camera  furnished  by  the  Bausch  &  Lomb 
Optical  Company.  {From  the  15th  edition 
I  /S96)  of  their  Catalog. ) 


EXPERIMENTS  IX  PHOTO-MICROGRAPHY. 

§  334-  The  following  experiments  are 

introduced  to  show  practically  just  how 
otie  would  proceed  to  make  photo-micro- 
graphs with  various  powers,  and  be 
reasonably  certain  of  fair  success.  If 
one  consults  prints  or  the  published 
figures  made  directly  from  photo-micro- 
graphs it  will  be  seen  that,  excepting 
the  bacteria,  the  magnification  ranges 
mostly  beween  10  and  150  diameter^-. 
The  technical  difficulties  in  making  g(  k  ■<! 
photo-micrographs  of  animal  tissues  at 
a  greater  magnification  are  so  great  that, 
while  they  may  be  used  as  the  basis  for 
'  figures,  they  are,  in  most  cases  not  suit- 
able for  direct  reproduction. 
§  335.  Photo-Micrographs  at  a  Magnification  of  5  to  20  Di- 
ameters.— In  the  study  of  embryology  and  the  morphology  of  small 
animals  or  of  individual  organs  like  the  brain,  it  is  frequently  desirable 
to  make  pictures  of  the  whole  object  in  its  natural  setting.  These  ob- 
jects and  their  surroundings  are  frequently  from  one  to  two  centimeters 
in  diameter,  that  is  of  a  size  too  great  to  be  satisfactorily  photographed 
with  microscopic  objectives.  In  common  with  other  observers  tin 
writer  has  found  the  short  focus,  wide  angled,  photographic  objectives 
to  give  excellent  results.  It  is  necessary  to  have  considerable  length 
I  i*2  to  2  meters)  of  bellows  for  this,  if  a  magnification  of  [0  to  15 
diameters  is  desired. 

Put  the  objective  in  position  in  the  front  of  the  camera  and  place  the 
object  on  some  kind  of  support  near  the  objective.     A   T-shaped   board 
13 


l94  PHOTO-MICROGRAPHY.  \CH.   VIII. 

with  a  hole  of  proper  size  is  good.  Then  with  a  glass  chimney  on  the 
lamp,  place  the  bull's  eye  between  the  lamp  and  object  (Fig.  157).  Put 
a  piece  of  white  paper  over  the  object  and  mutually  arrange  lamp  and 
bull's  eye  till  a  sharp  image  of  the  flame  may  be  seen  on  the  paper 
covering  the  object  (Fig.  157)-  Remove  the  paper  from  the  object 
and  proceed  to  focus.  This  is  accomplished  by  sliding  the  whole 
camera  toward  or  away  from  the  object,  see  also  §  339.  One  must  also 
shorten  or  lengthen  the  bellows  to  get  the  picture  of  the  proper  size. 

In  case  the  whole  specimen  is  not  illuminated  the  lamp  must  be 
turned  so  that  the  broad  side  of  the  flame  is  toward  the  object.  The 
mutual  arrangement  of  lamp  and  bull's  eye  must  also  be  such  that  the 
brightest  part  of  the  flame  illuminates  the  object.  If  the  bull's  eye 
is  moved  a  little  toward  the  lamp,  the  flame  will  be  sufficiently  broad- 
ened. It  must  be  remembered,  however,  that  the  best  and  most 
intense  illumination  is  obtained  when  the  object  appears  in  the  image 
of  the  flame.  When  the  object  is  evenly  illuminated  and  the  focus  is 
made  as  perfect  as  possible,  a  small  diaphragm  is  used  in  the  objective 
1  '•  e-tf  32  or  64)  and  the  plate  holder  with  the  sensitive  plate  is  put  in 
place  of  the  focusing  screen.  Some  kind  of  cover  is  put  over  the  ob- 
jective, the  slide  of  the  plate  holder  is  withdrawn  and  then  the 
objective  is  uncovered.  With  instantaneous,  orthochromatic  or 
isochromatic  plates  the  exposure  in  most  cases  need  not  be  over  30  to 
90  seconds,  depending  on  the  object  and  the  diaphragm. 

It  sometimes  occurs  that  the  whole  object  cannot  be  satisfactorily 
lighted.  In  that  case  one  may  use  diffused  day-light  as  follows  : 
Elevate  the  camera  so  that  the  object  is  against  the  clear  sky  for  a 
back  ground.  If  any  of  the  earth  should  form  part  of  the  back  ground 
that  part  of  the  object  would  not  be  sufficiently  illuminated.  It  is  best 
not  to  use  any  mirror.  The  light  from  the  sky  will  evenly  and  com- 
pletely illuminate  even  the  largest  object. 

Instead  of  elevating  the  camera  one  might  use  a  large  reflector 
covered  with  very  white  cloth  or  paper  and  set  at  an  angle  of  45  de- 
grees. This  reflector  would  then  serve  for  back  ground  equally  with 
the  clear  sky. 

§  336.  Focusing  Screen  for  Photo-Micrography.  One  cannot 
expect  a  picture  sharper  than  the  image  seen  on  the  focusing  screen. 
Hence  the  greatest  care  must  be  taken  in  focusing.  The  general 
focusing  may  be  done  with  the  unaided  eye  and  on  the  ground  glass, 
but  for  the  final  focusing  a  clear  screen  and  a  focusing  glass  must  be 
used  (Fig.  150). 


I  7/     /'///  ] 


PHOTO  MICROGRAPHY. 


Fig.  149.   Zeiss'  Apochromaticx  Projection   Objective  of  ~<< 
mm.  equivalent  focus,  for  photo-micography.    ■  Zeisss'  fatal 

Nc.  30 1,     This,  and  another  of  3$  mm.  foi  ns,  are  desig 
making  pictures  of  moderate  magnification.     Usually  rather 
large  objects  are  photographed  with  them.     Theobjecf  maybe  il- 
luminated in  the  ordinary  way.  They  are  used  without  an  o*  ular, 
like  a  photographic  objective.     The  one  of  jj  mm.  >ed 

into  the  tube  of  the  microscope  tike  an  ordinary  objective,  but 
the  one  of  70  mm.  here  shown,  is,  bv  means  of  a  conical 
adapter,  screwed  into  the  ocular  end  of  the  tube. 

/■'or  illuminating  the  object,  any  suitable  light  may  be  ;■ 
but  it  is  recommended  that  the  light  be  concentrated  by  me  . 
of  a  bull's  eye  or  some  form  of  combination  like  the  engraving  glass,  and  that  the 
condenser  be  so  placed  that  it  focuses  the  light  upon  the  objective,  not  upon  the 
object.     The  object  is  then  illuminated  with  a  converging  cone  of  light. 

For  the  clear  screen,  Mr.  Walmsley  and  others  have  recommended 
that  a  pencil  mark  or  cross  be  made  in  the  center  of  the  ground  glass 
and  then  that  a  large  circular  or  square  cover-glass  be  put  on  the 
ground  glass  with  Canada  balsam.  To  do  this,  warm  the  ground  glass 
carefully,  add  a  drop  of  rather  thick  balsam  to  the  center  on  the  ground 
side,  then  apply  the  cover  and  press  it  down  firmly.  After  the  balsam 
has  cooled  it  may  be  cleaned  off  around  the  cover  with  xylene  or  alcohol. 
The  balsam  will  fill  up  the  inequalities  in  the  glass  and  being  of  about 
the  same  refractive  power  will  make  this  part  of  the  glass  clear  as  if  it 
were  unground  (Fig.  150). 

For  using  the  focusing  glass  first  carefully  adjust  it  so  that  the  pencil 
cross  in  the  center  of  the  ground  glass  is  in  the  best  possible  focus. 
The  image  when  in  the  best  focus  must  then  be  in  the  same  plane  as 


Fig.  150.  Focusing  screen  with  clear  center 
for  the  final  adjustment  with  a  focusing  glass 
like  that  shown  in  Fig.  153  or  154. 

1 .  The  ground  surface  of  the  focusing  screen . 
it  is  translucent  but  not  transparent. 

2.  Central  clear  part  of  the  screen  made  by 
cementing  a  cover-glass  to  the  ground  surface 
with  Canada  balsam.  In  the  center  is  shown 
the  pencil  mark  to  indicate  the  plane  to  which 
the  focusing  glass  should  be  adjusted. 


1 

2 

X 

the  ground  side  of  the  focusing  screen.      If  the   uncovered  part  of  the 
focusing  screen  is  too  opaque,  rub  some  fine  oil  on  it  ;  only  a  little 


19" 


PHO  TO-MICROGRA  PH  Y. 


\CH.    VIII. 


should  be  used.  The  focusing  screen  as  thus  prepared  with  a  clear 
center,  serves  both  for  the  general  focusing  and  the  finest  focusing,  and 
avoids  the  danger  of  using  a  double  screen.  That  is,  the  fewer  the 
processes  the  less  the  liability  to  error. 


Fig.  151.  Peri^raphic  Objective  of  about  go  millimeters  equivalent  focus  for 
making  photo-micrographs  at  a  magnification  of  2  to  75  diameters.  It  was  found 
to  give  the  best  results  zvhen  used  right  end  to  as  in  ordinary  photography.  (From 
the  Gundlach  Optical  Co. ) 

FIG.  152.  Zeiss'  Anas- 
tig  mat  Objective  of  about 
S5  millimeters  equivalent 
focus  for  photo-micro- 
graphs at  a  magnification 
of  2  to  15  diameters.  Ex- 
cellent results  ivere  ob- 
tained with  this,  and  the 
other  short  focused  anas- 
tigmats.  On  the  whole 
the  results  were  more  sat- 
isfactory when  the  lens 
was  used  right  end  to  as 
in  ordinary  photography. 
(From  the  Bausch  and 
Lotnb  Optical  Co.) 

§  337.   Development  of  the  Negative.  — After  the  exposure,  comes 
the  development  ;  this  in  photo-micrography  requires  more  judgment 


CH.   17//.] 


I'UOlOMlih'OCk'.lI'llY. 


than  for  ordinary  photography.  The  ordinary  negative  is  liable  to 
have  too  much  contrast,  but  this  is  rarely  the  case  with  photo  micro 
graphs.  Any  good  developer  may  be  used.  One  can  as  a  rule  do  no 
better  than  to  follow  the  directions  accompanying  the  plates  used. 
The  writer's  experience  has  been  so  satisfactory  with  Mr.  Walmsley's 
developers  that  he  desires  to  call  attention  to  them.  The  develop<  i 
are  easily  made,  will  develop  anything  that  can  be  developed  and  one 
can  feel  confident  that  if  the  negative  is  not  good  the  fault  does  not  lie 
with  the  developer. 

The  best  photo-micrographic  negatives  made  by  the  writer  were  made 
with  Cnuner's  instantaneous  isochromatic  plates,  and  the  image  com- 
menced to  appear  with  Walmsley's  developer  in  :?'_>  to  3  minutes  and 
the  development  was  completed  in  12  to  15  minutes.  Excellent  nega 
tives  have  been  developed  in  less  time  and  also  when  it  required  half 
an  hour  to  develop  them.  The  temperature  has  much  to  do  with  the 
development  of  correctly  exposed  negatives,  so  that  no  rule  can  be 
given.  Metol  and  rodinal  developers  have  also  given  excellent  results 
and  in  a  shorter  time. 

If  one  desires  the  best  possible  results  it  is  necessary  to  avoid  the 
light  in  developing.  Even  the  ruby  light  of  the  dark  room  should  be 
avoided  as  much  as  possible,  for  the  plates  used  are  made  purposely 
sensitive  to  the  longer  rays  of  the  spectrum  (§  193).  After  the  nega- 
tive is  developed  and  washed  it  should  not  be  taken  to  the  light  till  it 
is  fixed.  Too  much  light  after  development  and  before  fixing,  injures 
the  clearness  of  the  negative. 


Fig.  153.  Focusing  Glass.     "  It  is  achromatic,  con- 
sisting of a  double  convex  crown  lens  and  a  negative 

meniscus  flint  lens  cemented  together"  //  screws 
into  the  brass  tube  and  is  thus  adjustable,  enabling 
one  to  focus  the  pencil  mark  in  the  dear  area  of  the 
focusing  screen  [Fig.  150)  with  great  accuracy.  It  also 
serves  to  focus  the  image  with  ease  and  accuracy. 
The  eye  must  not  be  too  close  to  the  upper  end  of  the 
focusing  glass  or  the  field  will  be  restricted.  {From 
the  Gundlach  Optical  Co. ) 


§338.   Labeling  and  Care  of  Negatives. — The  care,  printing,  etc., 

of  negatives  is  like  that  of  ordinary  negatives.      It  is  well  to  labi  1  them 


198 


PI  10  TO  MICROGR  A  PHY. 


\CH.    VIII. 


carefully,  however.  This  label  should  contain  as  far  as  possible,  the 
following  information  :  (i)  Kind  of  plate  ;  (2  )  Time  of  exposure  ;  (3) 
I. -lit  used  ;  (4)  Objective;  (5)  Ocular  ;  (6)  Magnification  ;  (7)  Name 
of  object  and  number  of  preparation  from  which  taken  ;  (8)  Date  of 
making  the  negative. 

Fig.  154.  Tripod  magnifier.  This  serves  fairly  well 
as  a  focusing  glass,  but  is  inferior  to  the  one  shown  in 
Fig.  /Si- 

$  339.  Low  Microscopic  Objectives. —  Mi- 
croscopic objectives  of  35  mm.  and  greater  focal 
length  may  be  attached  to  the  camera  directly 
after  the  manner  of  a  photographic  objective  if 
one  has  a  plate  made  with  the  proper  screw  for 
the  objective.  The  only  difficulty  is  in  focusing 
the  object  properly.  If  one  has  the  special  stand 
made  by  the  Bausch  and  Lomb  Optical  Co.,  or  something  equivalent  so 
that  the  object  may  be  moved  with  a  fine  screw,  the  focusing  can  be 
well  done.  Such  an  arrangement  is  also  very  convenient  in  using  the 
photographic  objectives  (Figs.  151-132)  and  the  apochromatic  objec- 
tives of  35  and  70  mm.  focus  made  by  Zeiss. 

Fig.  155.  Engraving  glass  to  seize  as 
a  bull's-eye  condenser.  {From  the 
Bausch  and  Lomb  Optical  Co. ). 

§  340.  Photo-Micrographs  of 
20  to  50  Diameters. — For  these, 
low  objectives  are  used  (35  mm.  to 
20  mm.  focus).  They  are  attached 
to  the  camera  as  described  in  §  339 
or  a  microscope  is  used.  If  the 
microscope  is  used  the  object  is 
placed  on  the  stage  and  focused  in 
the  usual  wav.  The  mirror  is  pref- 
erably removed  or  swung  aside  and  the  lamp  and  bull's  eye  mutually 
arranged  to  give  an  image  of  the  flame  as  described  in  §  335.  If  the 
object  is  small  the  achromatic  condenser  may  be  used  (See  §  342)  or 
one  of  the  Steinheil  magnifiers.  When  the  light  is  satisfactory  as  seen 
through  an  ordinary  ocular,  remove  the  ocular. 

(A)  Photographing  without  an  Ocular,  -After  the  removal  of  the 
ocular  put  in  the  end  of  the  tube  a  lining  of  black  velvet  to  avoid  re- 
flections. Connect  the  microscope  with  the  camera,  making  a  light 
tight  joint  and  focus  the  image  on  the  focusing  screen.       It  will  be 


(//.  /■///.] 


/ '//( >  7X  )  M I  CROC R A  /•//)■ 


necessary   to    focus   down    considerably   to   make-    the    image    clear. 
Lengthen  or  shorten  the  bellows  to  make  the  image  of  the  desired  siz 

then  focus  with  the  utmost  care.  Iu  case  the  field  is  too  much 
restricted  on  account  of  the  tube  of  the  microscope,  remove  tin-  draw- 
tube.  When  all  is  in  readiness  it  is  well  to  wait  for  three  to  five 
minutes  and  then  to  see  if  the  image  is  still  sharply  focused.  If  it  has 
got  out  of  focus  simply  by  standing,  a  sharp  picture  could  not  be  ob- 
tained. If  it  does  not  remain  in  focus,  something  is  faulty.  When  the 
image  remains  sharp  after  focusing  make  the  exposure.  From  15  to 
60  seconds  will  usually  be  sufficient  time  with  instantaneous  plates  and 
the  light  as  described. 


Fig.  156.  Lens  holder  composed  of  several  links  and  balls,  thus  giving  the  flexi- 
bility of  a  chain  and  enabling  one  to  turn  (he  lens  in  any  desired  direction.     Each 
link  has  a  screzv  to  take  tip  zvcar,  thus  insuring  permanence.      The  lens  is  grasped 
by  two  movable  pieces  -which  open  widely  enough  to  take  a  tripod  or  an  engraving 
glass  ;  they  also  hold  with  equal  facility  a  very  much  smaller  lens.      This  is  one  of 
the  most  efficient  and  satisfactory  lens  holders  ever  made.     It  is  epecially  good 
ladding  a  lens  for  concentrating  the  light  on   the   mirror  in   artificial  lighting 
(From  the  Bausch  &  Lomb  Optical  Co.) 

(B)  Photographing  with  a  Projection  Ocular. — If  the  object  is  small 
enough  to  be  included  in  the  field  of  a  projection  ocidar,  one  may  put 
that  in  place  of  the  ordinary  ocular.  The  first  step  is  then  to  focus  the 
diaphragm  of  the  projection  ocular  sharply  on  the  focusing  screen. 
Bring  the  camera  up  close  to  the  microscope  and  then  screw  out  the 
eye-lens  of  the  ocular  a  short  distance.  Observe  the  circle  of  light  on 
the  focusing  screen  to  see  if  its  edges  are  perfectly  sharp.  If  not.  con- 
tinue to  screw  out  the  eye-lens  until  it  is.  If  it  cannot  be  made  sh.irp 
by  screwing  it  out  reverse  the  operation.     Unless  the  edges  ol  the  light 


200 


PI  10  TO-MICROGRAPH  Y. 


\_CH.    VIII. 


circle,  i.  e.,  the  diaphragm  of  the  ocular,  is  sharp,  the  resulting  picture 
will  not  be  satisfactory.  When  the  diaphragm  is  sharply  focused  on 
the  screen,  the  microscope  is  focused  exactly  as  though  no  ocular  were 
present,  that  is,  first  with  the  unaided  eye  then  with  the  focusing  glass  ; 
the  object  should  be  in  focus  in  the  beginning. 


Fig.  157.  Arrangement  for  Artificial  Illumination. 

1.  Lamp  with  metal  chimney,  easily  made  by  rolling  up  some  ferrotype  plate 
and  making  a  slit-like  opening  in  one  side.  This  opening  should  be  covered  by  an 
oblong  cover-glass.  A  glass  slide,  being  of  considerable  thickness,  breaks  too 
easily.  The  lamp  should  have  a  wick  about  30  mm.  wide,  so  that  the  thickness  of 
the  flame,  if  taken  edgewise,  will  give  an  intense  light.  A  ivide  flame  also  enables 
one  to  get  a  larger  image  of  the  flame,  and  thus  illuminate  a  larger  object  than  as 
though  a  small  flame  were  used. 

2.  Bull's-eye  condenser  on  a  separate  stand.  The  engraving  glass  shown  in  Fig. 
755-  or  the  tripod  magnifier  {Fig.  154)  answers  fairly.  The  Steinheil  lenses  are 
still  better. 

3.  Screen  showing  image  of  the  flame  inverted. 

The  lamp  and  bulls-eye  stand  are  on  blocks  with  screw-eyes  as  leveling  screws. 

The  exposure  is  also  made  in  the  same  way,  although  one  must  have 
regard  to  the  greater  magnification  produced  by  the  projection  ocular 
and  increase  the  time  accordingly;  thus  when  the  X  4  ocular  is  used, 
the  time  should  be  at  least  doubled  over  that  necessary  when  no  ocular 
i->  employed. 

Zeiss  recommends  that  when  the  bellows  have  sufficient  length  the 
lower  projection  oculars  be  used,  but  with  a  short  bellows  the  higher 
ones.  It  is  also  sometimes  desirable  to  limit  the  size  of  the  field  by 
putting  a  smaller  diaphragm  over  the  eye  lens.  This  would  aid  in 
making  the  field  uniformly  sharp. 


CH   17//.]  PHOTOMICROGRAPHY.  201 

?j  341,  Photo- Micrographs  at  a  Magnification  of  100  to  150  Di- 
ameters.-—For  this,  the  simple  arrangements  given  in  the-  preceding 
section  will  answer,  but  the  objectives  must  be  of  shorter  focus,  8  to  3 

111111.      It  is  better,  however,  to  use  an  achromatic  condenser  instead  of 
the  engraving  glass  or  the  Steinheil  lens. 

$  342.  Lighting  for  Photo-Micrography  with  Moderate  and 
High  Powers. — (  100  to  2,500  diameters).  No  matter  how  good  oik's 
apparatus,  successful  photo-micrographs  cannot  be  made  unless  the  ob 
ject  to  be  photographed  is  properly  illuminated.  The  beginner  van  do 
nothing  better  than  to  go  over  with  the  greatest  care  the  directions  for 
centering  the  condenser,  for  centering  the  source  of  illumination,  and 
the  discussion  of  the  proper  cone  of  light  and  lighting  the  whole  field, 
as  given  on  pp.  391046.  Then  for  each  picture  the  photographer 
must  take  the  necessary  pains  to  light  the  object  properly.  An  achro 
matic  condenser  is  almost  a  necessity  (§76).  Whether  a  color-screen 
should  be  used  depends  upon  judgment  and  that  can  be  attained  only 
by  experience.  In  the  beginning  one  may  try  without  a  screen,  and 
with  different  screens  and  compare  results. 

A  plan  used  by  many  skillful  workers  is  to  light  the  object  and  the 
field  around  it  well  and  then  to  place  a  metal  diaphragm  of  the  proper 
size  in  the  camera  very  close  to  the  plate  holder.  This  will  insure  .1 
clean,  sharp  margin  to  the  picture.  Of  course  this  metal  diaphragm 
must  be  removed  while  focusing  the  diaphragm  of  the  projection 
ocular,  as  the  diaphragm  opening  would  be  smaller  than  the  image  of 
the  ocular  diaphragm. 

If  the  young  photo-micrographer  will  be  careful  to  select  for  his  first 
trials  objects  of  which  really  good  photo-micrographs  have  already 
been  made,  and  then  persists  with  each  one  until  fairly  good  results  an- 
attained,  his  progress  will  be  far  more  rapid  than  as  if  poor  pictures  of 
many  different  things  were  made.  He  should,  of  course,  begin  with 
low  magnifications. 

§  343.  Adjusting  the  Objective  for  Cover-Glass. — After  the  ob 
ject  is  properly  lighted,  the  objective,  if  adjustable,  must  be  corrected 
for  the  thickness  of  cover.  If  one  knows  the  exact  thickness  of 
the  cover  and  the  objective  is  marked  for  different  thickness,  it 
is  easy  to  get  the  adjustment  approximately  correct  mechanically, 
then  the  fine  or  final  correction  depends  on  the  skill  and  judg- 
ment of  the  worker.  It  is  to  be  noted  too  that  if  the  objective  is 
to  be  used  without  a  projection  ocular  the  tube  length  is  practically  ex 
tended  to  the  focusing  screen  and  as  the  effect  of  lengthening  the  tube 
is  the  same  as  thickening  the  cover-glass,   the  adjusting    collar    must 


2o: 


/'//( )  Tl  )■  MICROGRAPHY. 


\CH.   VIII. 


be  turned  to  a  higher  number  than  the  actual  thickness  of  the  cover 
calls  for    see  §  96). 

^  344.  Photographing  "Without  an  Ocular. — Proceed  exactly  as 
described  for  the  lower  power,  but  if  the  objective  is  adjustable  make 
the  proper  adjustment  for  the  increased  tube-length. 

ij  345.  Photographing  with  a  Projection  Ocular. — Proceed  as  de- 
scribed in  £  343,  only  in  this  case  the  objective  is  not  to  be  adjusted 
for  the  extra  length  of  bellows.  If  it  is  corrected  for  the  ordinary 
ocular,  the  projection  ocular  then  projects  this  correct  image  upon  the 
focusing  screen. 


Frc  15S.  Zeiss'  Vertical  Photo-micro- 
graphic  Camera.  A.  Set  scre?a  holding  the 
rod  (S)  in  any  desired  position.  P,  O.  Set 
screws  by  which  the  bellows  are  held  in  place. 
B.  Stand  with  tripod  base  in  which  the  sup- 
porting rod  (S)  is  held.  This  rod  is  now 
graduated  in  centimeters  and  is  a  ready 
means  of  determining  the  length  of  the  cam- 
era. M.  Mirror  of  the  microscope.  L.  The 
sleeve  serving  to  make  a  light  tight  connection 
between  the  camera  and  microscope.  O.  The 
lower  end  of  the  camera.  A'.  The  upper  end 
of  the  camera  where  the  focusing  screen  and 
plate  holder  are  situated.  [From  Zeiss'  Photo- 
micrographic  Catalog). 


§  346.  Determination  of  the  Magnification  of  the  Photo-Micro- 
graph.— After  a  successful  negative  has  been  made,  it  is  desirable  and 
important  to  know  the  magnification.  This  is  easily  determined  by 
removing  the  object  and  putting  in  its  place  a  stage  micrometer.  If 
now  the  distance  between  two  or  more  of  the  lines  of  the  micrometer 
is  obtained  with  dividers  and  the  distance  measured  on  one  of  the  steel 
rules  the  magnification  is  obtained  by  dividing  the  size  of  the  image  by 
the  known  size  of  the  object  (§  146).      If  now  the  length   of  the  bel- 


c 


o 


en.  i  v//. ; 


PHOTO-MIi  ROGRAPHY. 


Pigs.  160  r6i.  Fine  tint,  half-tone  reproductions  of  photo-micrograph 
made  by  Mrs.  Gage,  to  show  the  possibilities  of  photo-micrograph}   with  pho 
graphic  objectives  and  with  /<>.v  m  'croscopic  objectives  -without  a  proje  turn 

i .  Frontal  section  of  the  head  of  a  large  red  Di  'my  tylus  viridescens  I  red  m 
at  the  level  oftheportae  of  the  brain,  magnified  10  diameters.     Negative  made 
with  a  Gundlach  perigraphic  objective  of  about  go  mm.  equivalent  focus. 

2.  Frontal  section  of  a  tana!  Diemyctylus  about  10  millimeters  in  length. 
Negative  made  with  a  Winkel  objective  of  22  millimeters  equivalent  focus;  no 
ocular.  Magnified  s°  diameters.  By  the  permission  of  Mrs.  Susanna  Phelps 
Cage,  from  the  Wilder  Quarter  Century  Hook. 

lows  from  the  tube  of  the  microscrope  is  noted,  say  <>n  a  record  table 
like  that  in  section  348,  one  can  get  a  very  close  approximation  to  the 

power  at  some  other  time   by   using  the  same  optical  combination  and 
length  of  bellows. 

FlG.  1.59.   Rack  for  drying  negatives.     (From 
the  Rochester  Optical  Co. ) 

$  347.  Photo-Micrographs  at  a  Mag- 
nification of  500  to  2000  Diameters.— 
For  this  the  homogenous  immersion  objec- 
tives should  be  employed,  and  as  it  would 
require  a  long  bellows  to  get  the  higher 
magnification  with  the  objective  alone,  it  i> 
best  to  use  the  projection  oculars. 
For  this  work  the  directions  given  in  £  342  must  be  followed  with 
great  exactness.  The  edge  of  the  lamp  flame  will  be  sufficient  to  lil  1 
the  field  in  most  cases.  With  many  objects  the  time  required  with 
good  lamp  light  is  not  excessive  ;  viz.,  1  %  to  3  minutes.  The  reason 
of  this  is  that  while  the  illumination  diminishes  directly  as  the  square 
of  the  magnification,  it  increases  with  the  increase  in  numerical  aper- 
ture, so  that  the  illuminating  power  of  the  homogeneous  immersion  is 
great  in  spite  of  the  great  magnification  1  £  31). 

For  work  with  high  powers  a  stronger  light  than  the  petroleum  lamp 
is  employed  by  those  doing  considerable  photo- micrograph  v.  Very 
good  work  may  be  done,  however,  with  the  petroleum  lamp. 

It  may  be  well  to  recall  the  statement  made  in  the  beginning,  that 
the  specimen  to  be  photographed  must  be  of  especial  excellence  for  all 
powers.      Xo  one  will  doubt  the  truth  of  the  statement  who  undertal, 
to  make  photo-micrographs  at  a  magnification  of  500  to  2         liameters. 


204 


/  '//( )  TO- MICROGRAPHY. 


{CH.    VIII. 


I'll..! 

pher, 

r. 

o 

f 

si 

u 

Remarks 

itive 
Good. 

Developer. 

Rodinal 

i  -31 . 

11 

4-1 
CO 

s 

Cramer's 

Inst.  Iso- 

chromatics 

1-  = 

0  11 

0  i 
O  o 

■J 
0 

'3 

4J 

Silver 
and  Hema- 
toxylin. 

1 

u 

11 

0 

Silvered 

1 1.  patic 

Ligt. 

Neclurus. 

Kxposure 

and 
Remarks. 

V 

V 

(fl 

0 

O 
0 

Light, 
Hour, 

Date. 

Daylight. 

10 

~  0 

1 

Cond. 

1.    - 

0  y 

<  - 

M  ignifica- 

ti.m  and 

Length  of 
Bellows. 

0 

■1. 

i  = 

Ocular. 

■* 

X 

0 
u 

'? 

u 

- 

Camera 

and 
Objective. 

•-    r. 

11 

ill.   VIII.]  PHOTOMICROGRAPHY 

PHOTOGRAPHING     NATURAL     HISTORY    SPECIMENS     WITH    A    VERTICAL 

C  \M1.K  \ 

£  349.  For  most  natural  history  specimens  it  is  inconvenient,  and  for 
many  impossible,  to  use  a  horizontal  camera  and  to  raise  the  objects 
in  a  vertical  position.     In  order  to  have  the  objects  horizontal  either  a 

mirror  must  be  used  or  preferably  the  camera  itself  maj  be  so  arranged 
that  it  may  be  put  in  a  vertical  position. 

For  the  List  twenty  years  such  a  camera  lias  been  in  use  in  the  Ana- 
tomical Department  of  Cornell  University  for  photographing  all  kinds 
of  specimens;  among  these,  fresh  brains,  and  hardened  brains  have 
been  photographed  without  the  slightest  injury  to  them.  Furthermore, 
as  many  specimens  are  so  delicate  that  they  will  not  support  their  own 
weight,  they  may  be  photographed  under  alcohol  or  water  with  a  ver 
tical  camera  and  the  result  will  be  satisfactory  as  a  photograph  and 
harmless  to  the  specimen. 

A  great  field  is  also  open  for  obtaining  life  like  portraits  of  water 
animals.  Freshly  killed  or  etherized  animals  are  put  into  a  vessel  of 
water  with  a  contrasting  back  ground  and  arranged  as  desired  then 
photographed.  The  fins  have  something  of  their  natural  appearance 
and  the  gills  of  branchiate  salamanders  float  out  in  the  water  in  a 
natural  way.  In  case  the  fish  tends  to  float  in  the  water  a  little  mer- 
cury injected  into  the  abdomen  or  intestine  will  serve  as  ballast. 

The  photographs  obtainable  in  water  are  almost  if  not  quite  as  sharp 
as  those  made  in  air.  Even  the  corrugations  on  the  scales  of  such 
fishes  as  the  sucker  |  Catostomus  teres  1  show  with  great  clearness.  In- 
deed so  good  are  the  results  that  excellent  fine  tint,  half  tone  plates 
may  be  produced  from  the  pictures  thus  made,  also  excellent  photo- 
gravures. In  those  cases,  as  in  anatomical  preparations,  where  the 
photograph  rarely  answers  the  requirements  of  a  scientific  figure,  >ti!l 
a  photograph  serves  as  a  most  admirable  basis  for  a  scientific  figure. 
The  photograph  is  made  of  the  desired  size  and  all  the  parts  are  in 
correct  proportion  and  in  the  correct  relative  position.  From  this 
photographic  picture  may  be  traced  all  the  outlines  upon  the  drawing 
paper,  and  the  artist  can  devote  his  whole  time  and  energj  t>>  giving  the 
proper  expression  without  the  tedious  labor  of  making  measurements. 

"  While  the  use  of  photograph)'  for  outlines  as  bases   for   figures  di- 

*Papers  on  this  subject  were  given  by  the  writer  at  the  meeting  of  the  American 
Association   for  the  Advancement  of  .Science  in    1S79,  and  ;it  the  meeting  of  the 
.Society  of  Naturalists  of  the  eastern   United  States  in    1883 ;  ami  in  Science,  v 
III,  pp.  4.13.  441 


206  PHOTOMICROGRAPHY.  \_CH.    VIII. 

minishes  the  labor  of  the  artist  about  one-half  it  increases  that  of  the 
preparator;  and  herein  lies  oik- of  its  chief  merits.  The  photographs 
being  exact  images  of  the  preparations,  the  tendency  will  be  to  make 
them  with  greater  care  and  delicacy,  and  the  result  will  be  less  imagi- 
nation and  more  reality  in  published  scientific  figures;  and  the  objects 
prepared  with  such  care  will  be  preserved  for  future  reference." 

"  In  the  use  of  photography  for  figures  several  considerations  arise  : 
i°.  The  avoidance  of  distortion  ;  2°.  The  adjustment  of  the  camera  to 
obtain  an  image  of  the  desired  size;  30.  Focusing;  40.  Fighting  and 
centering  the  object  ;  50.  Obtaining  outlines  for  tracing  upon  the 
drawing-paper." 

"  i°.  While  the  camera  delineates  rapidly,  the  image  is  liable  to  dis- 
tortion. I  believe  opticians  are  agreed,  that,  in  order  to  obtain  correct 
photographic  images,  the  objective  must  be  properly  made,  and  the 
plane  of  the  object  must  be  parallel  to  the  plane  of  the  ground  glass. 
Furthermore,  as  most  of  the  objects  in  natural  history  have  not  plane 
surfaces,  but  are  situated  in  several  planes  at  different  levels,  the  whole 
object  may  be  made  distinct  by  using  in  the  objective  a  diaphragm  with 
a  small  opening." 

"  20.  By  placing  the  camera  on  a  long  table,  and  a  scale  of  some  kind 
against  the  wall,  the  exact  position  of  the  ground  glass  for  various 
sizes  may  be  determined  once  for  all.  These  positions  are  noted  in 
some  way   (on   the  brass  guide,    3,   in   the   apparatus    here    figured). 

Whenever  it  is  desired  to  photograph  an  object,  natural  size,  for  ex- 
ample, the  ground  glass  is  fixed  in  the  proper  position  indicated  on  the 
brass  guide  (Fig.  162,  3).  Then  as  the  relative  position  of  the  objec- 
tive and  the  ground  glass  must  not  be  varied,  it  is  necessary,  in  focus- 
ing, to  move  the  camera  toward  or  away  from  the  object,  or  the  re- 
verse. To  do  this,  the  camera  is  fastened  to  a  board  which  moves  in  a 
frame  by  means  of  a  screw  Fig.  162,  7.  Whenever  the  camera  is  to  be 
moved  considerably, — as  to  a  position  for  twice  natural  size  from  one 
giving  an  image  of  half  natural  size, — the  position  of  the  camera  on 
the  board  is  changed  by  loosening  the  two  thumb-screws  clamping  it  to 
the  movable  board  (Fig.  162,  5,  6).  The  approximate  position  for  the 
various  sizes  being  once  determined  and  noted,  it  is  but  a  moment's 
work  to  set  the  camera  for  any  enlargement  or  reduction  within  its 
range." 

30.  The  object  is  placed  on  a  horizontal  support,  and  so  arranged 
that  the  lighting  will  give  prominence  to  the  parts  to  be  especially 
emphasized.  For  a  contrasting  background,  black  velveteen  for  light, 
and  white  paper  for  dark,  objects,  have  been  found  excellent. 


C/I    17//.] 


PHOTO-MICROGRAPHY. 


6 


Fig.  162. 

Arrangement  of  a  vertical  or  horizontal  camera  for  making  photographs;  of  nat- 
ural history  objects — sectional  view. 

1.  The  photographic  objective.  It  should  be  of  the  best  quality  and  rectilineal 
so  that  there  may  be  no  distortion. 

2.  Cone  to  increase  the  length  of  the  camera  and  avoid  shadows. 

3.  Graduated  rod  to  support  the  front  of  the  camera  and  hold  it  rigid,  and  also 
to  serve  as  guide  to  the  various  magnifications  and  reductions  most  commonly 
desired. 

./.  (hound  glass.  It  is  an  advantage  to  haze  a  clear  space  in  the  middle  for 
accurate  focusing,  as  for  the  photo-micrographic  camera  [fig".  153  . 

5.  Camera  bed  fastened  to  the  sliding  focusing  board,  6. 

6.  Focusing  board  to  which  the  camera  is  clamped. 

7.  Focusing  screw. 

8.  Solid  piece  connecting  the  focusing  board  and  focusing 

9.  Hinge  on  which  the  camera  swings. 

10.  Drawer  in  which  are  kept  the  objective  and  other  accessor  ies. 
//.  Box  of  sand  or  other  heavy  material  to  serve  as  ballast. 
The  large  screw  eyes  in  the  legs  of  the  table  serve  as  lev. -ling  sere 

4°.  If  the  photographic  prints  are  to  be  used  solely  t"<>r  outlines,  the 
well-known  bine  prints  so  much  used  in  engineering  and  architecture 


2o8  PHOTO-MICROGRAPHY.  [CH.   VI U. 

may  be  made.  If,  however,  light  and  shade  and  fine  details  are  to  be 
brought  out  with  great  distinctness,  either  an  aristotype,  platinotype  or 

a  bromide  print  is  preferable.  In  whatever  way  the  print  is  made,  it 
is  blacked  on  the  back  with  soft  lead-pencil,  put  over  the  drawing- 
paper,  and  the  outlines  traced. 

OUTLINES    OBTAINED    DIRECTLY    BY    MEANS    OF    THE  CAMERA. 

?;  350.  While  it  is  desirable  to  make  photographs  of  objects  in  many 
cases,  this  may  frequently  be  avoided  and  a  tracing  made  directly  from 
the  camera.  The  object  is  arranged  as  for  a  ph  .tograph  and  well 
lighted.  Then  the  ground  glass  is  changed  for  a  plain  glass  and  the 
tracing  paper  is  put  over  the  glass.  If  the  head  and  screen  are  covered 
in  some  way  the  image  may  be  seen  and  traced  very  easily.  This  will 
o-ive  a  reversed  image,  but  that  is  easily  remedied  by  turning  the  paper 
over  in  making  the  tracing  on  the  drawing  paper. 

Frequently  also  one  wishes  to  enlarge  or  reduce  a  drawing  or  outline 
already  made.  If  the  figure  or  outline  is  placed  in  a  good  light  the 
image  may  be  made  of  any  desired  size  and  either  photographed  or 
traced  as  just  described.  Tracings  of  any  desired  size  may  also  be  ob- 
tained of  negatives,  but  for  this  one  must  employ  the  method  of  light- 
ing described  in  §  335,  where  the  clear  sky  is  taken  for  a  background 
and  illuminant.  Of  course  artificial  light  may  also  be  used,  but  it  is 
less  satisfactory  unless  one  has  abundant  facilities. 

An  excellent  method  of  making  large  diagrams  is  to  use  a  photo- 
graphic objective  and  project  the  image  into  a  dark  room,  something 
after  the  manner  of  a  stereopticon,  then  one  can  trace  the  image  directly 
on  the  material  on  which  the  diagram  is  to  be  drawn. 

jj  351.  Prints  of  Photo-Micrographs  and  Mechanical  Printing. 
After  one's  negatives  are  made,  prints  from  them  may  be  obtained 
from  a  photographer,  but  from  the  author's  experience,  unless  the  pho- 
tographer is  familiar  with  the  kind  of  printing  necessary  to  bring  out 
the  features  most  desired,  the  results  will  not  be  very  satisfactory.  It 
is  better  to  make  one's  own  prints,  and  this  can  be  very  easily  done 
with  the  excellent  aristotype  and  platinotype  paper  on  the  market.  It 
may  also  be  well  done  with  the  bromide  paper.  The  last  has  the  advant- 
age of  being  capable  of  furnishing    prints  by  lamp  as  well  as  daylight. 

For  mechanical  prints,  half  tones  and  photogravures,  one  should  get 
first  as  good  a  negative  as  possible.  And  for  photogravures  the  so- 
called  stripping  plate  should  be  used  so  that  the  picture  will  not  be  re- 
versed.     For  half  tones  the  print  should   have  a  good   deal  of  contrast 


CH.   17//.]  PHOTOMICROGRAPHY. 

and  some  pure  white.     Witb  good  printing  the  fine  half  tone  work  on 
the  larger  natural  history  specimens  is  capable  of  giving  very  ^at; 
tory  results  as  may  be  seen  by  observing  Fig.  [60  and  [61. 

For  the  methods  of  Photo-Micrography  and  Photography  in  general,  consult  the 
winks  mentioned  in  the  bibliography.     Especial  attention   is  also  called  t<>  the 
papers  of  Woodward  "  On  an   improved   method   of  photographing  histological 
preparations  by  sunlight,    1871,   and  011  the  Application  of  Photography  t<>  M 
crometry." 

To  the  papers  and  discussions  of  A.  C.  Mercer  in  the  various  volumes  of  the 
Proceedings  of  the  American  Microscopial  Society. 

To  the  papers  and  discussions  on  photomicrography  by  I'illard  ;  Journal  of  the 
Royal  Micr.  Soc.  1^92,  and  1V93,  also  in  the  Amer.  Jour.  Med.  Sciences  1893.  The 
paper  by  Dr.  G.  A.  Piersol, — "Some  experiences  in  Photo-Micrography"  in  the 
American  Annual  of  Photography  for  1890  will  also  be  found  very  instrui  live  and 
helpful.  In  every  volume,  and  freqeuntly  in  every  number,  of  the  microscopical 
journals  of  this  and  foreign  countries  one  may  find  articles,  notes  or  referent  es  to 
work  in  photo  microgaphy.  Excellent  papers  are  also  frequently  found  in  the 
photographic  journals  and  annuals. 

Thomas  J.  Wray. — Photo-Micrography  by  use  of  ordinary  objectives,  practically 
considered  with  specimens  of  work.  Proc.  Amer.  Micr.  Soc.  Vol.  XVIII,  (1S96K 
The  specimens  in  connection  with  this  paper  impressed  one  that  simple  apparatm 
was  no  bar  to  success. 

Sunlight  and  Heliostat. — In  case  one  wishes  to  utilize  sunlight  for  Photo- 
Micrography  it  would  he  of  great  advantage  to  consult  the  papers  of  Woodward 
mentioned  above  and  also  the  paper  of  Mercer  in  the  Jour.  Roy.  Micr.  Soc,  1S92, 
pp.  305  to  318.  For  work  with  sunlight,  a  heliostat  is  almost  a  necessity.  An  ex- 
cellent and  inexpensive  form  was  devised  by  Dr.  L,.  Deck  and  described  and 
figured  in  the  Proc.  Amer.  Micr.  Soc,  1S91,  pp.  49-50.  A  good  form  of  heliostat 
is  also  figured  and  described  in  Van  Heurck,  pp.  265-266. 

Sunlight  is  not  so  easily  managed  as  some  form  of  artificial  light,  even  when 
one  possesses  a  heliostat. 

For  samples  of  photo-micrographic  work,  see  the  magnificent  photo  prints  ex- 
hibited by  Dr.  Woodward  at  the  Centennial  Celebration  at  Philadelphia  in  1" 
the  works  of  Mason,  Sternberg,  Piersol,  Wilson  and  Starr;  Carpenter-Dallinger, 
Pringle,  Bousfield  Nehauss,  etc.,  besides  the  great  embryological  and  inorphe- 
logical  journals  where  photo-micrographic  plates  are  frequently  published. 
Catalogs  of  photo-micrographic  apparatus,  in  some  cases,  have  admirable  speci- 
mens of  photo-micrographs. 


14 


APPENDIX. 


Abbe's  Test-Plate;  The  Apertometer  ;  Experimental  Detei  ruination  of  the 
Equivalent  Focus  of  the  Objective  and  of  the  Ocular;  Testing  Homogeneous 
Fluid  ;   Preparation  of  Diagrams  ;  Drawings  for  Photo-Engraving. 

\  352.  For  the  sake  of  those  who  may  have  opportunity  to  use  Abbe's  Test- 
Plate  the  following  directions  from  Zeiss  are  appended  : 

"CARL   ZEISS,   JENA.      ON   THE   METHOD   OF   USING    ABBE'S   TEST-PLATE." 

"  This  test-plate  is  intended  for  the  examination  of  objectives  with  reference  to 
their  corrections  for  spherical  and  chromatic  aberration  and  for  estimating  the 
thickness  of  the  cover  glass  for  which   the  spherical  aberration  is  best  corrected." 

"  The  test-plate  consists  of  a  series  of  cover-glasses  ranging  in  thickness  from 
0.09  mm.  to  o  24  mm.,  silvered  on  the  under  surface  and  cemented  side  by  side  on 
a  slide.  The  thickness  of  each  is  written  on  the  silver  film.  Groups  of  parallel 
lines  are  cut  through  the  film  and  these  are  so  coarsely  ruled  that  they  are  easily 
resolved  by  the  lowest  powers,  yet  from  the  extreme  thinness  of  the  silver  they 
form  a  very  delicate  test  for  objectives  of  even  the  highest  power  and  widest 
aperture.  To  examine  an  objective  of  large  aperture  the  plates  are  to  be  focused 
in  succession  observing  each  time  the  quality  of  the  image  in  the  center  of  the 
field  and  the  variation  produced  by  using  alternately  central  and  very  oblique 
illumination.  When  the  objective  is  perfectly  corrected  for  spherical  aberration 
for  the  particular  thickness  of  cover-glass  under  examination,  the  contour  of  the 
lines  in  the  center  of  the  field  will  be  perfectly  sharp  by  oblique  illumination 
without  any  nebulous  doubling  or  indistinctness  of  the  minute  irregularities  of  the 
edges.  If  after  exactly  adjusting  the  objective  for  oblique  light  central  illumina- 
tion is  used  no  alteration  of  the  adjustment  should  be  necessary  to  show  the  con- 
tours with  equal  sharpness." 

'  If  an  objective  fulfills  these  conditions  with  any  one  of  the  plates  it  is  free 
from  spherical  aberration  when  used  with  cover-glasses  of  that  thickness  ;  on  the 
other  hand  if  every  plate  shows  nebulous  doubling  or  an  indistinct  appearance  of 
the  edges  of  the  silver  lines,  with  oblique  illumination,  or  if  the  objective  requires 
a  different  adjustment  to  get  equal  sharpness  with  central  as  with  oblique  light, 
then  the  spherical  correction  is  more  or  less  imperfect." 

"  Nebulous  doubling  with  oblique  illumination  indicates  overcorrection  of  the 
marginal  zone,  want  of  the  edges  without  marked  nebulosity  indicates  undercor- 
rection  of  this  zone;  an  alteration  of  the  adjustment  for  oblique  and  central 
illumination,  that  is,  a  difference  of  plane  between  the  image  in  the  peripheral 
and  central  portions  of  the  objective  points  to  an  absence  of  concurrent  action  of 
the  separate  zones,  which  may  be  due  to  either  an  average  under  or  overcorrection 
or  to  irregularity  in  the  convergence  of  the  rays." 

'  The  test  of  chromatic  correction  is  based  on  the  character  of  the  color  bands, 
which  are  visible  by  oblique  illumination.  With  good  correction  the  edges  of  the 
silver  lines  in  the  center  of  the  field  should  show  but  narrow  color  bands  in  the 
complementary  colors  of  the  secondary  spectrum,   namely  on  one  side  yellow-. 


API  ENDIX.  ]  .  /  BBE'S  TEST-PL  A  '//■.'.  2 1 1 

green  to  apple-green  011  the  other  violet  to  rose.      The  more  perfect  the  correi  tion 
of  1  he  spherical  aberration  the  clearer  this  color  band  appears." 

"  To  obtain  obliquity  of  illumination  extending  to  the  marginal  Eoneof  the  ob- 
jective and  a  rapid  interchange  from  oblique  to  central  light  Abbe's  [Uuminatiug 
apparatus  is  very  efficient,  as  it  is  only  necessary  t<>  move  the  diaphragm  in  use 
nearer  to  or  further  from  the  axis  by  the  rack  and  pinion  provided  for  the  purpose. 
For  the  examination  of  immersion  objectives,  whose  aperture  as  a  rule  is  greater 
than  iN<>0  in  air  ami  those  homogeneous  immersion  objectives,  which  considerably 
exceed  this,  it  will  be  necessary  to  bring  the  under  surface  of  the  1  est  plate  into 
contact  with  the  upper  lens  of  the  illuminator  by  means  of  a  drop  of  water,  gly- 
cerin or  oil." 

"  In  this  case  the  change  from  central  to  oblique  light  may  be  easily  effect,  d  by 
the  ordinary  concave  mirror  but  with  immersion  lenses  of  large  aperture  it  is  im- 
possible to  reach  the  marginal  zone  by  this  method,  and  the  best  elTect  has  to  be 
searched  for  after  each  alteration  of  the  direction  of  the  mirror." 

"  For  the  examination  of  objectives  of  smaller  aperture  (less  than  4o°-50°)  we 
may  obtain  all  the  necessary  data  for  the  estimation  of  the  spherical  and  chro- 
matic corrections  by  placing  the  concave  mirror  so  far  laterally,  that  its  edge  is 
nearly  in  the  line  of  the  optic  axis  the  incident  cone  of  rays  then  only  filling  one- 
half  of  the  aperture  of  the  objective.  The  sharpness  of  the  contours  and  the 
character  of  the  color  bands  can  be  easily  estimated.  Differences  in  the  thickness 
of  the  cover-glass  within  the  ordinary  limits  are  scarcely  noticeable  with  such  ob- 
jectives." 

"  It  is  of  fundamental  importance  in  employing  the  test  as  above  described  to 
have  brilliant  illumination  and  to  use  an  eye-piece  of  high  power." 

"  When  from  practice  the  eye  has  learnt  to  recognize  the  finer  differences  in  the 
quality  of  the  contour  images  this  method  of  investigation  gives  very  trustworthy 
results.  Differences  in  the  thickness  of  cover-glasses  of  0.01  or  0.02  mm.  can  be 
recognized  with  objectives  of  2  or  3  mm.  focus." 

"  With  oblique  illumination  the  light  must  always  be  thrown  perpendicularlv  to 
the  direction  of  the  lines."  # 

"  The  quality  of  the  image  outside  the  axis  is  not  dependent  on  spherical  and 
chromatic  correction  in  the  strict  sense  of  the  term.  Indistinctness  of  the  con- 
tours towards  the  borders  of  the  field  of  view  arises  as  a  rule,  from  unequal  111.! 
nification  of  the  different  zones  of  the  objective;  color  bands  in  the  peripheral 
portion  (with  good  color  correction  in  the  middle)  are  caused  by  unequal  magnifica- 
tion of  the  different  colored  images." 

"Imperfections  of  this  kind,  improperly  called  'curvature  of  the  field,'  are 
shown  to  a  greater  or  less  extent  in  the  best  objectives,  where  the  aperture  is  con- 
siderable." 


FlG.  164.   Set  0/  lines  under  our  >  glass  in  the  Abbe   Test  Plate. 


212 


A  PER  TO  METER. 


[APPENDIX. 


DETERMINATION  OF  THE  APERTURE  OF  OBJECTIVES. 
§  353.  Determination  of  the  Aperture  of  Objectives  with  an  Apertometer. — Ex- 
cellent directions  for  using  the  Abbe  apertometer  maybe  found  in  the  Jour.  Roy. 
Micr.  Soc,  [878,  p.  19,  and  18S0,  p.  20;  in  Dippel,  Zimmerman  and  Czapski.  The 
following  directions  are  but  slightly  modified  from  Carpenter-Dallinger,  pp  337- 
138.  The  Abbe  apertometer  involves  the  same  principle  as  that  of  Tolles,  but  it 
is  carried  out  in  a  simpler  manner  ;  it  is  shown  in  Fig.  165.     As  seen  by  this  figure 


Fig.  165.  Abbe  Apertometer. 
it  consists  of  a  semi-circular  plate  of  glass.  Along  the  straight  edge  or  chord  the 
glass  is  beveled  at  450,  and  near  this  straight  edge  is  a  small,  perforated  circle,  the 
perforation  being  in  the  center  of  the  circle.  To  use  the  apertometer  the  micro- 
scope is  placed  in  a  vertical  position,  and  the  perforated  circle  is  put  under  the  mi- 
croscope and  accurately  focused.  The  circular  edge  of  the  apertometer  is  turned 
toward  a  window  or  plenty  of  artificial  light  so  that  the  whole  edge  is  lighted. 
When  the  objective  is  carefully  focused  on  the  perforated  circle  the  draw-tube  is 
removed  and  in  its  lower  end  is  inserted  the  special  objective  which  accompanies 
the  apertometer.  This  objective  and  the  ocular  form  a  low  power  compound  mi- 
croscope, and  with  it  the  back  lens  of  the  objective,  whose  aperture  is  to  be  meas- 
ured, is  observed.  The  draw-tube  is  inserted  and  lowered  until  the  back  lens  of 
the  objective  is  in  focus.  "In  the  image  of  the  back  lens  will  be  seen  stretched 
across,  as  ft  were,  the  image  of  the  circular  part  of  the  apertometer.  It  will  ap- 
pear as  a  bright  band,  because  the  light  which  enters  normally  at  the  surface  is  re- 
flected by  the  beveled  part  of  the  chord  in  a  vertical  direction  so  that  in  reality  a 
fan  of  l8o°  in  air  is  formed.  There  are  two  sliding  screens  seen  on  either  side  of 
the  apertometer  ;  they  slide  on  the  vertical  circular  portion  of  the  instrument. 
The  images  of  these  screens  can  be  seen  in  the  image  of  the  bright  band.  These 
screens  should  now  be  moved  so  that  their  edges  just  touch  the  periphery  of  tin- 
back  lens.  They  act,  as  it  wrere,  as  a  diaphragm  to  cut  the  fan  and  reduce  it,  so 
that  its  angle  just  equals  the  aperture  of  the  objective  and  no  more."  "This 
angle  is  now  determined  by  the  arc  of  glass  between  the  screens  ;  thus  we  get  an 
angle  in  glass  the  exact  equivalent  of  the  aperture  of  the  objective.  As  the  nu- 
merical apertures  of  these  arcs  are  engraved  on  the  apertometer  they  can  be  read 
off  by  inspection.  Nevertheless  a  difficulty  is  experienced,  from  the  fact  that  it  is 
not  easy  to  determine  the  exact  point  at  which  the  edge  of  the  screen  touches  the 
periphery  of  the  back  lens,  or  as  we  prefer  10  designate  it,  the  limit  of  aperture, 
for  curious  as  the  expression  may  appear  we  have  found  at  times  that  the  back 
lens  of  an  objective  is  larger  than  the  aperture  of  the  objective  requires.  In  that 
case  the  edges  of  the  screen  refuse  to  touch  the  periphery." 

In  determining  the  aperture  of  homogeneous  immersion  objectives  the  proper 
immersion  fluid  should  be  used  as  in  ordinary  observation.  So,  also,  with  glycerin 
or  water  immersion  objectives. 


APPENDIX.]        TESTER  FOR  IMMERSION  LIQUID. 


TESTING    HOMOGENEOUS    IMMERSION    LIQUID. 

§354.  [n  order  Lhat  one  shall  realize  the  full  benefit  of  the  homogeneous  im- 
mersion principle  it  is  necessary  that  the  homogeneous  immersion  liquid  should 
be  truly  homogeneous.  In  order  that  the  ordinary  worker  ma)  be  able  to  tc^t  the 
liquid  used  by  him,  Professor  Hamilton  I..  Smith  devised  a  •  om posed  of  a 

slip  of  glass  in  which   was  ground  accurately  a  small  concavity  and  another  1 
fectly  plain  slip  to  act  as  cover.     (See  Proc.  Amer.  Micr.  Soc,  1  It 

will  he  readily  seen    that   this  concavity,  if  tilled  with   air  or  any  liquid  "l"  | 
tractive  index  than  glass,  will  act  as  a  concave  or  dispersing  lens.      If  filled   with   a 
liquid  of  greater  refractive  index  than  glass,  the  concavity  would  act  like  a  <  om 
lens,  hut  if  filled  with  a  liquid  of  the  same  refractive  index  as  glass,  that  is,  liquid 
optically  homogeneous  with  glass,  then  there  would  he  no  effect  whatever. 

In  using  this  tester  the  liquid  is  placed  in  the  concavity  and  the  cover  put 
This  is  hest  applied  by  sliding  it  over  the  glass  with  the  concavity.  A  small 
amount  of  the  liquid  will  run  between  the  two  slips,  making  optical  contact  on 
both  surfaces.  One  should  he  careful  not  to  include  air  bubbles  in  the  concavity. 
The  surfaces  of  the  glass  are  carefully  wiped  so  that  the  image  will  not  be  ob- 
scured. An  adapter  with  society  screw  is  put  on  the  microscope  and  the  objective 
is  attached  to  its  lower  end.  In  this  adapter  a  slot  is  cut  out  of  the  rij^ht  width 
and  depth  to  receive  the  tester  which  is  just  above  the  objective.  As  object  it  is 
well  to  employ  a  stage  micrometer  aud  to  measure  carefully  the  diameter  of  the 
field  without  the  tester,  then  with  the  tester  far  enough  inserted  to  permit  of  the 
passage  of  rays  through  the  glass  but  not  through  the  concavity,  aud  finally  the 
concavity  is  brought  directly  over  the  back  lens  of  the  objective.  This  can  be 
easily  determined  by  removing  the  ocular  and  looking  down  the  tube. 

Following  Professor  Smith's  directions  it  is  a  good  plan  to  mark  in  some  way  the 
exact  position  of  the  tube  of  the  microscope  when  the  micrometer  is  in  focus 
without  the  tester,  then  with  the  tester  pushed  in  just  far  enough  to  allow  the  light 
to  pass  through  the  plane  glass  and  finally  when  the  light  traverses  the  concavity. 
The  size  of  the  field  should  be  noted  also  in  the  three  conditions  :;  . 

\  355  The  following  table  indicates  the  points  with  a  tester  prepared  by  the 
Guudlach  Optical  Co.,  and  used  with  a  16  mm.  apochromatic  objective  of  Zeiss,  X4 
compensation  ocular,  achromatic  condenser,  1.00  N.  A.     Fig.   \\  1  : 


TESTER    AND    LIQUID    IN    THE 
CONCAVITY. 


NT3  tester  used 

Whole  thickness  of  the  tester  at 
one  end.  not  over  the  cavity  .  . 

Tester  with  air  in  the  cavity  .  .    . 

Tester  with  water 

Tester  with  u^%  alcohol        ... 

kerosene  .... 

Guudlach  Opt.  Co  's 

horn,  liquid    ... 

Bausch  &  LfOmb  Opt.  Co. 'shorn, 
liquid 

Leitz'  bom.  liquid       

Zeiss'  bom.  liquid 


SIZE  OF  THE 
FIELD. 


KI.I-.V  \TI()N    OF    1  111-.    1  I   l:i 
1  SSARY  TO  RESTORE  THE 

M  ><  1  s. 


[.825  mm.     .     .Standard  position. 

1.85  mm.  .    .     No  change  of  focus. 

.6  mm.     .    .     Tube  raised  6  mm. 

1.075  mm.  3'..  nun. 

1. 15  mm ,;  mm. 

1    1  mm 2  nun. 


.825  nun. 


mm. 


mm. 


1  825  mm.     .         ... 

r.825  nun ,•'.',  mm 

1.825  mm ,    .  mm 


214       DETERMINA  TION  OF  EQUIVALENT  FOCUS.     [APPENDIX. 

It  will  be  seen  by  glaucing  at  the  above  table  that  whenever  the  liquid  in  the 

tester  is  of  lower  index  than  glass,  that  the  concavity  with  the  liquid  acts  as  a 
concave  lens,  or  in  other  words  like  an  amplifier  (\  152),  and  the  field  is  smaller 
than  when  no  tester  is  used.  It  will  also  be  seen  that  as  the  liquid  in  the  con- 
cavity approaches  the  ^lass  in  refractive  index  that  the  field  approaches  the  size 
when  no  tester  is  present.  It  is  also  plainly  shown  by  the  table  that  the  greater 
the  difference  in  refractive  index  of  the  substance  in  the  concavity  and  the  glass, 
the  more  must  the  tube  of  the  microscope  be  raised  to  restore  the  focus. 

If  a  substance  of  greater  refraction  than  glass  were  used  in  the  tester  the  field 
would  be  larger,  i.  e  ,  the  magnification  less,  and  one  would  have  to  turn  the  tube 
down  instead  of  up  to  restore  the  focus.  The  tester  used  in  these  experiments 
was  made  by  the  Gundlach  Optical  Company  of  Rochester. 

EQUIVALENT  FOCUS  OF  OBJECTIVES  AND  OCULARS. 

§  356.  To  work  out  in  proper  mathematical  form  or  to  ascertain  experimentally 
the  equivalent  foci  of  these  complex  parts  with  real  accuracy  would  require  an 
amount  of  knowledge  and  of  apparatus  possessed  only  by  an  optician  or  a  physicist. 
The  work  may  be  done,  however,  with  sufficient  accuracy  to  supply  most  of  the 
needs  of  the  working  microscopist.  The  optical  law  on  which  the  following  is 
based  is: — "  The  size  of  object  and  image  varies  directly  as  their  distance  from 
the  center  of  the  lens.'" 

By  referring  to  Figs.  14,  16,  21,  it  will  be  seen  that  this  law  holds  good.  When 
one  considers  compound  lens-systems  the  problem  becomes  involved,  as  the  centre 
of  the  lens  systems  is  not  easily  ascertainable  hence  it  is  not  attempted,  and  only 
an  approximately  accurate  result  is  sought. 

\  357-  Determination  of  Equivalent  Focus  of  Objectives. — Look  into  the  upper 
end  of  the  objective  and  locate  the  position  of  the  back  lens.  Indicate  the  level 
in  someway  on  the  outside  of  the  objective.  This  is  not  the  center  of  the  object- 
ive but  serves  as  an  arbitrary  approximation.  Screw  the  objective  into  the  tube 
of  the  microscope.  Remove  the  field  lens  from  a  micrometer  ocular,  thus  making 
a  positive  ocular  of  it  (Fig.  21).  Pull  out  the  draw-tube  until  the  distance  between 
the  ocular  micrometer  and  the  back  lens  is  250  millimeters.  Use  a  stage  microm- 
eter as  object  and  focus  carefully.  Make  the  lines  of  the  two  micrometers 
parallel  (Fig.  101).  Note  the  number  of  spaces  on  the  ocular  micrometer  required 
to  measure  one  or  more  spaces  on  the  stage  micrometer.  Suppose  the  two  microm- 
eters are  ruled  in  ,',,  mm.  and  that  it  required  10  spaces  on  the  ocular  micrometer 
to  enclose  2  spaces  on  the  stage  micrometer,  evidently  then  5  spaces  would  cover 
one.  The  image,  A'R1  Fig.  21  in  this  case  is  five  times  as  long  as  the  object,  A,B. — 
Now  if  the  size  of  object  and  image  are  directly  as  their  distance  from  the  lens  it 
follows  that  as  the  size  of  object  is  known  ( -j-(1-  mm.),  that  of  the  ima»e  directly 
measured  ( J  %  mm. ),  the  distance  from  the  lens  to  the  image  also  determined  in  the 
beginning,  there  remains  to  be  found  the  distance  between  the  objective  and  the 
object,  which  will  represent  approximately  the  equivalent  focus.  The  general 
formula  is,  Object,  O  :  Image,  I  :  :  equivalent  focus,  F  :  250.  Supplying  the 
known  values,  0  =  T2,j,  I  =  {';,  then  r25  in  :  1  mm  :  :  F  :  250  whence  F  =  50111m.  That 
is,  the  equivalent  focus  is  approximately  50  millimeters. 

§  358.  Determination  of  Initial  or  Independent  Magnification  of  the  Objective. 
The  initial  magnification  means  simply  the  magnification  of  the  real  image  (A1  B1, 
Fig.  21)  unaffected  by  the  ocular.     It  may  be  determined  experimentally  exactly 


.  XPPENDIX.1  PREP.  IRA  '/'/<  W  OF  />/.  XGRA  \fS  2  1  5 

as  described  in  \  357.  For  example,  the  image  of  the  object  mm.  measured 
by  the  ocular  micrometer,  at  a  distance  of  250  mm.  is  mm.,  i.  e.t  it  is  five  times 
magnified,  hence  the  initial  magnification  of  the  50  mm.  objective  xi- 

mately  five. 

Knowing  the  equivalent  focus  of  an  objective,  one  can  determine  its  initial  mag- 
nification by  dividing  250  nun.  by  the  equivalent  focus  in  millimeters.  Thus  the 
initial  magnification  of  a  5  nun.  objective  is  50;   of  a  ;  mm.,  ;  of 

a  2  nun.,  - '■':"       125,  etc. 

§  359  Determining  the  Equivalent  Focus  of  an  Ocular.— If  one  knows  the  ini- 
tial magnification  of  the  objective  (§  358)  the  approximate  equivalent  focus  of  the 
ocular  can  be  determined  as  follows  : 

The  field  lens  must  not  be  removed  in  this  case.  The  distance  between  the  | 
tion  of  the  real  image,  a  position  indicated  in  the  ocular  by  a  diaphragm,  and  the 
back  lens  of  the  objective  should  be  made  250  mm.,  as  described  in  \  357,  358,  then 
by  the  aid  of  Wollaston's  camera  lucida  the  magnification  of  the  whole  microscope 
is  obtained,  as  described  in  \  149.  As  the  initial  power  of  the  objective  is  known, 
the  power  of  the  whole  microscope  must  be  due  to  that  initial  power  multiplied  by 
the  power  of  the  ocular,  the  ocular  acting  like  a  simple  microscope  to  magnify  the 
real  image  (Fig.  21 ). 

Suppose  one  has  a  50  mm.  objective,  its  initial  power  will  be  approximately  5. 
If  with  this  objective  and  an  ocular  of  unknown  equivalent  focus  the  magnifica- 
tion of  the  whole  microscope  is  50,  then  the  real  image  or  initial  power  of  the  ob- 
jective must  have  been  multiplied  10  fold.  Now  if  the  ocular  multiplies  the  real 
image  10  fold  it  has  the  same  multiplying  power  as  a  simple  lens  of  25  mm.  focus 
for,  using  the  same  formula  as  before  ;o  5  :i  —  50  :  :  f  :  250  whence  F  =  25.  The 
matter  as  stated  above  is  really  very  much  more  complex  than  this,  but  this  gives 
an  approximation. 

For  a  discussion  of  the  equivalent  focus  of  compound  lens-systems,  see  modern 
works  on  physics  ;  see  also  C.  R.  Cross,  on  the  Focal  Length  of  Microscopic  Ob- 
jectives, Franklin  lust.  Jour.,  1S70,  pp.  401-402;  Monthly  Micr.  Jour.,  [870,  pp. 
149-159.     J.J.  Woodward,  on  the  Nomenclature  of  Achromatic  I  ves,  Anier. 

Jour.  Science,  1872,  pp.  406-414;  Monthly  Micr.  Jour.,  1S72.  pp.  66-74.  W.  S. 
Franklin,  method  for  determining  focal  lengths  of  microscope  lenses.  Physical 
Review,  Vol.  I.  [893,  p.  142.  See  pp.  1037  to  1049  of  Carpenter-Dalliuger  for 
mathematical  formulae;  also  Daniell,  Physics  for  medical  students;  Czapski, 
Theorie  der  optischen  Instrumente ;  Dippell,  Nageli  und  Schwendeuer,  Zitnmer- 
mann. 

PREPARATION    <>F    DIAGRAMS. 

$  360.  For  class  room  work  many  diagrams  are  needed.  Those  which  are  pur- 
chased soon  get  out  of  date,  but  from  their  cost  one  does  not  feel  like  throwing 
them  away.  It  is  a  fact,  however,  that  so  much  of  one's  education  comes  through 
the  eye  that  it  is  not  safe  to  have  an  incorrect  diagram  for  students  to  studj  The 
visual  impression  is  liable  to  outlive  the  verbal  correction.  To  avoid  incorrect  di- 
agrams or  those  that  no  longer  represent  the  present  state  of  knowledge  one  1 
use  blackboard  diagrams.  By  means  of  the  flexible,  or  roll  blackboards,  one  ma) 
make  the  diagrams  anywhere  ami  hang  them  in  the  lecture-room.  This  also  is  of 
advantage  where  several  must  use  the  same  lecture-room. 

For  permanent  diagrams  which  shall  be  as  easy  to  make  as  the  ordinary   black- 


2 1 6  DRA  U  INGS  FOR  I  'HO  TO-ENGRA  VING.       [APPENDIX. 

board  drawings  and  so  cheap  that  there  is  no  temptation  to  preserve  them  after  their 
usefulness  is  past,  one  may  adopt  the  suggestion  of  Dr.  Dunnington  of  the  Uni- 
versity of  Virginia  and  make  the  diagrams  on  manilla  paper  with  ordinary  black- 
board crayons,  and  then  fix  them  so  that  they  will  not  rub  by  hanging  them  where 
the  face  cannot  touch  and  putting  the  fixative  on  the  back  with  a  brush  or  a  sponge. 
The  fixative  may  be  readily  prepared  by  mixing  one  liter  (1000  cc. )  of  ordinary 
painter's  turpentine  with  ioo  cc.  of  dammar  varnish.  If  these  are  well  shaken  to- 
gether the  dammar  will  be  dissolved  in  the  turpentine,  and  then  if  the  mixture  is 
put  on  the  hack  of  the  diagram  it  will  soak  through  the  paper  and  upon  drying, 
will  fix  the  crayon  lines  so  that  they  will  not  rub.  At  the  present  day  black  black- 
board crayons  are  manufactured  and  they  are  somewhat  easier  to  shade  with,  and 
ver}-  much  cheaper  for  all  black  work  than  the  Conde  crayons.  The  Conde  cray- 
ons are  better  for  lettering,  however. 

It  requires  only  about  12  hours  for  the  fixative  to  dry.  Diagrams  ma}-  be  used 
in  less  time,  but  the}-  should  not  be  rolled  much  sooner.  If  one  wishes  to  have 
rollers,  and  they  are  very  convenient,  they  are  easily  made  by  using  what  the  car- 
penters call  "  half-round."  If  two  of  these  are  used,  the  paper  being  put  between, 
and  then  the  sticks  nailed  together,  very  neat  looking  diagrams  are  produced.  Of 
course  if  it  is  desired,  water  colors  may  be  used  upon  these  diagrams  either  before 
or  after  the  crayon  has  been  fixed. 

In  making  diagrams  from  figures  in  books,  if  one  desires  to  enlarge  a  definite 
number  of  times  the  drawing  paper  should  be  laid  out  in  squares.  If  these  are 
made  lightly  in  pencil  they  will  not  show  in  the  finished  diagram  unless  one  scans 
it  closely. 

DRAWINGS    FOR    PHOTO-ENGRAVING. 

(WRITTEN    BY    MRS.    GAGE). 

\  361.  The  inexpensive  processes  of  reproducing  drawings  bring  within  the 
reach  of  every  writer  upon  scientific  subjects  the  possibility  of  presenting  to  the 
eye  by  diagrams  and  drawings  the  facts  discussed  in  the  text.  Though  artistic 
ability  is  necessary  for  perfect  representation  of  an  object,  neatness  and  care  will 
enable  any  one  to  make  a  simple  illustrative  drawing,  from  which  an  exact  copy  is 
obtained  and  a  plate  prepared  for  printing. 

\  362.  A  shaded  drawing  prepared  by  washes  of  India  ink  can  be  reproduced  by 
the  "  half-tone  process,"  which  is  the  same  as  that  used  in  the  case  of  photographs. 
(See  \  351,  Figs.  160-161).  The  process  usually  called  photo  engraving  is  that  by 
which  all  the  line  drawings  in  this  book  were  reproduced,  and  is  much  less  expen- 
sive than  the  "half-tone"  process.  For  photo-engraving,  only  pure  black  and 
white  can  be  used  in  the  drawing,  as  shades  of  gray  are  not  successfully  repro- 
duced. 

\  363.  Outfit  for  Drawings.— A  perfectly  squared  drawing  board  ;  a  T-square  ; 
thumb  tacks  ;  a  right-line  pen  ;  a  circle  pen  ;  an  assortment  of  smooth  pointed 
pens,  including  for  fine  work  a  lithographic  pen  ;  very  soft,  hard,  and  medium 
pencils;  fine  scissors;  fine  forceps;  a  sharp  pointed  knife  or  scalpel;  smooth, 
white  bristol  board  ;  perfectly  black  ink.  To  test  the  ink  draw  extremely  fine 
lilies  and  look  at  them  with  a  magnifying  glass.  Most  of  the  water-proof  and 
liquid  India  inks  on  the  market  answer  well. 

2  364.  Size  of  Drawing. — It  is  first  necessary  to  decide  upon  the  scale  at  which 
the  drawings  are  to  be  made.     It  is  always  recommended  that  they  be  made  large, 


.  \PPENDIX.]       DRA  WINGS  FOR  PHOTi  >  /.\<.A'.  117  \ 

in  order  that  iu  tin-  photo  eugraving  they  may  be  reduced  in  si/c,  and  thus 
the  apparent  imperfections  of  the  drawings.     The  amount  of  reduction  in n-^t  be  ■ 
termined  by  each  individual,  depending  upon  whether  a  fine  and  * 

and  broad  style  is  natural.  In  the  lot  iner  case  a  reduction  of  '  *,  is  Buffii  ieiit  ;  in 
tin- latter,  'or  even  '  _.  is  desirable.  The  most  generally  useful  reduction  is  found 
to  be  ! .   . 

If  one  knows  the  size  of  the  page  upon  which  the  figure  or  plate  is  to  he  prillt<  d, 
it  is  easy  to  plan  exactly  as  to  the  size  <>f  the  drawings.  For  example,  a  finished 
plate  is  to  be  iox  16  cm.,  and  the  reduction  is  • , ,  then  the  drawing  should  he  made 
'_.  larger  than  the  plate  ;  that  is,  15  x  24  cm.  and  '  j  reduction  will  produce  the  1 
act  size  needed.  Then  the  enlarged  page  so  determined  can  he  outlined  by  the 
T-square  and  the  drawings  artistically  arranged  in  the  space.  If  one  does  not 
know  at  the  outset  the  size  of  the  page,  the  drawings  may  be  made  upon  -■ 
papers,  closely  trimmed,  arranged  on  card  hoard,  care  being  taken  that  they  do 
not  overrun  the  limit  of  the  page,  as  above  determined,  and  pasted  in  position. 
This  is  an  exceedingly  practical  method  of  procedure,  even  when  the  size  of  the 
page  is  known.  Care  should  be  taken  to  keep  all  straight  lines  representing  per- 
pendicular and  horizontal  directions  upon  the  individual  drawings,  in  correct  rela- 
tions to  the  corresponding  outlines  of  the  completed  plate.  For  instance,  the  scale 
of  magnification  (3  177)  should  be  parallel  with  the  bottom  of  the  page,  and  this  i-> 
easily  determined  by  the  T-square. 

?,  365.  For  mechanical  drawings,  the  exact  plan  is  carefully  plotted,  dots  are 
placed  for  intersections  and  endings  of  lines ;  the  lines  are  lightly  put  in  with  a 
sharp  pointed,  medium  pencil,  and  then  with  a  right-line  pen  and  a  circle  pen. 

\  366.  Vox  a  large  natural  history  object,  a  blue  print  \  349  or  a  tracing  nude 
from  a  camera  [\  350)  is  obtained  ;  or  for  a  microscopic  object,  a  camera  lucid. 1 
embryograph  drawing  is  made  ($  176,  178).  The  outlines  are  carefully  corrected 
by  comparison  with  the  object,  and  sharply  defined.  The  back  of  this  photograph 
or  drawing  is  blackened  with  a  soft  lead  pencil,  and  rubbed  gently  with  a  bit  of 
paper  to  make  an  even  coating.  To  transfer  the  drawing  it  is  placed  over  the 
drawing  paper  and  secured  in  the  desired  position  with  thumb  tacks.  All  the  out- 
lines are  then  traced  with  an  ivory  point,  or  a  very  hard,  round  pointed  lead  pen- 
cil, and  the  outlines  will  appear  on  the  drawing  paper.  These  are  again  lightly 
retouched  with  a  pencil  to  complete  defective  lines.  If  it  is  desired  to  reverse  a 
drawing,  as  in  the  case  of  a  tracing  made  with  the  camera  \\  350),  it  IS  placed 
against  a  well  lighted  window  pane,  its  outlines  followed  upon  the  back  with  a  pen- 
cil, and  then  its  original  outlines  retraced  with  a  soft  pencil.  It  is  placed  face  dow  11 
upon  the  drawing  paper  and  the  lines  upon  the  back  traced  with  a  hard  point,  and 
the  outlines  will  be  transferred  to  the  drawing  paper. 

A.  If  a  diagram  or  outline  drawing  is  desired,  the  outlines  thus   made  are  f>1 
lowed  by  the  pen,  heavy,  li^ht,  and  interrupted  lines  being  used  to  indicate  differ- 
ent classes  of  facts,  according  to  the  special  need. 

B.  If  a  shaded  drawing  is  to  be  made,  only  those  outlines  are  traced  ill  ink  which 
indicate  cut  edges  of  tissue,  or  for  some  reason  need  to  be  emphasized  in  an  espe- 
cial manner.  Then  the  shading  is  put  in,  the  deep  Bhadows  and  high  lights  being 
strongly  marked  and  the  gradations  carefully  determined.     This  is  more  easily 


In  any  case,  the  amount  of  reduction   should  be  clearly  indicated  10  the  ]>'■■ 
engraver. 


2 18  DRA  II 'INGS  FOR  PHOTO-EK<. RA  I  rING.       [APPENDIX. 

ilone  if  a  pencil  or  wash  drawing  is  first  made.  The  shading  may  either  be  done 
by  dotting  the  surface  with  a  pen  (stipple)  or  by  lines.  It  is  much  easier  to  pro- 
duce acceptable  results  by  stippling,  because  a  slight  erasure  may  be  made  or  a 
deeper  shadow  obtained  by  adding  a  few  carefully  placed  dots.  This  method  is 
well  adapted  for  showing  the  structure  of  histological  specimens. 

C.  If  lines  are  to  be  used  for  shading,  the  beginner  will  find  it  more  satisfactory 
to  use  the  fine  lithographic  pen,  the  line  extending  from  the  shadow  toward  the 
high  light  and  ending  in  a  series  of  dots.  The  deeper  shadow  is  reinforced  by  a 
second  or  even  third  series  of  lines,  and  these  should  rarely  cross  each  other  at 
right  angles.  They  should  rather  follow  the  contour  of  the  surface  represented 
and  in  crossing  should  form  diamond  shaped  spaces.  A  study  of  good  copper  or 
steel  engravings  will  aid  one  in  securing  a  proper  method  and  instances  of  excel- 
lent pen  work  abound  in  the  first-class  illustrated  magazines  from  which  valuable 
suggestions  can  be  obtained 

1).  It  is  sometimes  desirable  to  be  able  to  put  white  lines  ever  a  dark  shadow. 
In  this  case  a  white  ink  may  be  used. 

K.  Occasionally  a  very  dark  picture  is  needed.  In  this  case  a  specially  prepared 
paper  may  be  used  and  covered  with  an  even  wash  of  black  ink.  Lines  are  cut 
out  with  a  sharp  instrument,  thus  leaving  wdiite  lines  on  a  black  background. 

P.  Paper  with  a  raised  stipple  is  sometimes  used.  Upon  this  the  shading  is  done 
by  wax  crayons,  the  crayon  adhering  to  the  elevations  and  leaving  the  depressions 
white.  Lines  and  deep  shadows  are  put  in  with  ink.  In  this  way  more  excellent 
drawings  can  be  made  than  by  the  more  laborious  method  with  pen  and  ink,  but 
as  a  rule  the  results  of  the  photo  engraving  are  not  so  satisfactory. 

G.  In  case  it  is  necessary  to  show  different  colors,  the  drawing  is  made  in  dif- 
ferent colored  inks  exactly  as  desired,  pale  colors  being  avoided.  The  photo  en- 
graver makes  a  plate  for  each  color,  and  the  accuracy  necessary  in  their  production 
and  printing  makes  the  process  many  times  more  expensive  than  the  simple  repro- 
duction of  a  black  and  wdiite  drawing. 

\  367.  Lettering.  —  A  halftone  engraving  from  a  photograph  may  have  letters 
placed  upon  it  by  a  second  printing. 

The  most  carefully  prepared  drawing  may  be  artistically  ruined  by  placing  upon 
it  ill-formed  and  irregular  lettering,  and  as  even,  artistic  lettering  is  almost  a  trade 
by  itself,  it  is  recommended  that  either  the  drawing  be  sent  to  the  photo-engraver 
with  directions  to  have  the  letters  properly  put  in,  or  wdiat  is  more  satisfactory,  to 
have  the  letters,  abbreviations  and  words  needed,  printed  and  separately  placed  on 
the  drawings. 

To  do  this,  type  of  the  proper  size  for  the  reduc  ion  determined  upon  is  chosen. 
Numbers  of  the  following  size  have  been  found  convenient  for  numbering  the 
separate  figures  of  a  plate  for  '3  reduction  ; 

60,         61,         62, 

of  the  following  size  for  setial  parts  of  the  individual  drawings, 
1,  2,  3,  4,  5, 

while  italic  letters  of  the  following  size  show  what  has  proved  most  satisfactory  for 
Yl  reduction, 


APPENDIX.}       DRAWINGS  FOR  PHOTO-ENGRAVING.  2ig 

R.    be  L.  g.  t.     Filament  of  Necturus  C.  Leucocyh 

For  's  reduction  the  following  are  large  enough, 

Meten.        mtc.        >>i//>.        >////>>-.        /    2    3    /    5. 

[t  is  convenient  to  have  the  complete  alphabet  both  in  capitals  and  Bmall  letters 
and  series  of  numbers  in  different  sizes.  The  printing  should  1"-  done  on  firm,  but 
light  weight,  white  paper  (20  lb.  Demy  has  been  found  satisfactoi 

The  slips  thus  printed  an-  pinned  upon  a  hoard  and  coated  upon  the  back  with 
liquid  gelatin  {''.  3U  <  and  dried.  With  fine  scissors  the  words  are  cut  Out  neatly, 
moistened  and  with  fine  forceps  placed  upon  the  drawing  and  pressed  down  with 
blotting  paper.  Upon  a  large  and  complicated  plate,  the  letters  and  words  should 
be  placed  so  as  not  to  offend  the  eye.  Those  which  are  intended  to  be  parallel  to 
each  other  and  to  an  edge  of  the  plate  should  be  strictly  so,  as  determined  in  ad- 
justing thein  by  means  of  the  T-square.  The  numbers  of  the  different  figures  of 
a  plate  can  be  arranged  so  as  to  lend  to  the  general  harmony  and  still  be  k< 
close  to  the  designated  figure.  If  a  great  deal  of  lettering  and  numbering  is  ap- 
plied a  careful  plan  of  the  exact  place  of  each  word  must  be  determined  before 
any  are  fastened,  otherwise  confusion  results.  A  word  may  be  made  to  follow  the 
arc  of  a  circle,  by  snipping  the  slip  on  which  it  is  printed  with  the  scissors  and 
curving  it  after  it  is  moistened. 

\  36S.  After  the  letters  and  words  are  thus  fastened,  if  any  of  them  are  not 
exactly  upon  the  parts  to  which  they  refer,  a  line  from  the  part  to  the  word  should 
be  drawn  with  the  right  line  pen  and  T-square.  Dotted  or  full  lines  may  be  used, 
according  to  which  will  show  most  clearly — and  in  passing  over  a  deep  shadow, 
it  may  be  scratched  out  with  a  sharp  scalpel,  leaving  a  white  line. 

\  369.  After  and  only  after  every  part  of  the  drawing  is  as  complete  as  possible 
should  the  eraser  be  lightly  used  to  remove  pencil  marks  and  the  sharply  pointed 
knife  or  scalpel  to  remove  slight  defects,  as  the  overrunning  of  an  inked  line  or  to 
pick  out  shadows  which  are  too  deep.  This  is  to  avoid  roughening  the  surface  of 
the  drawing  paper  and  thus  making  it  impossible  to  add  clear  cut  lines  with  Un- 
pen. 

\  y,o.  Considerable  modification  of  a  photo- engraving  can  be  made  by  a  skillful 
engraver  with  his  tools.  Most  of  the  illustrations  of  the  current  magazines  are 
half  tone  or  photo-engravings  retouched  by  the  engraver.  Slight  defects  of  a  plate, 
a  line  a  trifle  too  long,  a  complete  line  which  should  be  interrupted,  a  superfluous 
line  can  be  easily  remedied,  but  a  line  cannot  easily  be  added.  A  deep  shadow 
can  be  lightened  or  removed,  but  it  is  cheaper  to  renew  the  drawing  than  to  under- 
take extensive  changes  in  the  plate.  In  the  case  of  a  plate  made  up  of  drawi: 
011  separate  papers,  the  edge  of  each  paper  casts  ,1  shallow  which  would  appear  in 
the  plate  except  that  it  is  cut  out  by  the  photo-engraver,  and  in  the  same  way  the 
edges  of  the  slips  used  for  lettering  cast  shadows  which  the  engraver  reniov<  8.  Ii 
any  such  lines  should  appear  in  the  proof  they  can  be  indicated  and  removed  be- 
fore used  in  printing. 


BIBLIOGRAPHY. 


The  books  and  periodicals  named  below  in  alphabetical  order  pertain  wholly  or  in  part  to  the 
microscope  or  microscopical  methods.  They  are  referred  to  in  the  text  by  recognizable  abbrevi- 
ations. 

For  current  microscopical  and  histological  literature,  the  Journal  of  the  Royal  Microscopical 
Society,  the  Index  Medicus,  the  Zoologischer  Anzeiger,  and  the  Zeitschrift  fiir  wissens:haftliche 
Mikroskopie,  Anatomischer  Anzeiger,  Biologisches  Centralblatt  and  Physiologisches  Central- 
blatt,  and  the  smaller  microscopical  journals  taken  together  furnish  nearly  a  complete  record. 

References  to  books  and  papers  published  in  the  past  may  be  found  in  the  periodicals  just 
named,  in  the  Index  Catalog  of  the  Surgeon  General's  library  ;  in  the  Royal  Society's  Catalog  of 
Scientific  Papers,  and  in  the  bibliographical  references  given  in  special  papers.  A  full  list  of  peri- 
odicals may  also  be  found  in  Vol.  XVI  of  the  Index  Catalog. 

BOOKS. 

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Anthony,  Win.  A.,  and  Brackett,  C.  F.— Elementary  text-book  of  physics.  7th  ed.  Pp.  527,  165 
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Barker. — Physics.     Advanced  course.     Pp.  902,  380  Fig      New  York,  1892. 

Bausch,  E-—  Manipulation  of  the  microscope.     Pp.  95,  illustrated.     Rochester,  1891. 

Beale,  L-  S. — How  to  work  with  the  microscope.  5th  ed.  Pp.  51S,  illustrated.  London,  1880. 
Structure  and  methods. 

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micographie,  comprenant  la  technique  et  les  applications  du  microscope  a  l'histologie  vegdtale, 
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Behrens,  W.,  Kossel,  A.,  und  Sehiefferdecker,  P. — Das  Mikroskop  uud  die  Methoden  der  mikro- 
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Brewster,  Sir  David.— A  treatise  on  the  microscope.  From  the  7th  ed.  of  the  Eucyc.  Brit.,  with 
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Brewster,  Sir  David. — A  treatise  on  optics.     Illustrated.     New  edition.     London,  1S53. 
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Carpenter,  W.  B. — The  microscope  and  its  revelations.  6th  ed.  Pp.  8S2,  illustrated.  London, 
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Carpenter-Dallinger. — The  microscope  and  its  revelations,  by  the  late  William  B.  Carpenter. 
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Foster,  Frank  P.— An  illustrated  encyclopaedic  medical  dictionary,  being  a  dictionary  of  th< 

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French  and  German  languages.     Illustrated  four  quarto  volume-      1-   --1893. 
Fraenkel  und  Pfeiflfer. — Atlas  der  Bacterieu-Kunde.     Berlin,  1889 
Francotte,  P. — Manuel  de  technique  microscopique,     Pp.  r.-    no  Fig      Brussels 

Francotte,  P. — Microphotographie  appliquee  a  l'histologie,  1'anatomie  compareeet  l'<  mbi 
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Frey,  K.— The  microscope  and  microscopical  technology.    Translated  and  edited  by  G    K  Cut- 
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63  Fig.      London  and  New  York,   [88o.      1  -93. 

Gibbs,  H. — Practical  histology  and  pathology.     Pp.  107.     London    1880.    Methods. 

Giltay,  Dr.  E. — Siebcn  Objcctc  unter  dem  Mikro-kop.      Kinfiihrung   in   die   Grundlehrei 
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Goodale,  G.  I..  Physiological  botany.    Pp    ;        |6,  illustrated.    New  York,  1885     Structure  and 
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Griffith  and  Henfrey. — The  Micrographic  Dictionary  ;  a  guide  to  the  examination  and  lnv»  sti 
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Gould,  G.  M.— The  illustrated  dictionary  of  medicine,  biology  and  alll<  -       IllaSl 

3d  ed  ,  Philadelphia,  1896.    This  is  recognized  as  the  best  single  volutn<  medical  diet 
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Halliburton,  W.  D.— A  text-book  of  chemical  physiology  and  pathologj      Pp  •  lllu* 

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222  BIBLIOGRAPHY. 

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224  BIBLIOGRAPHY. 

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Wethered,  M    -Medical  microscopy.     1 'p.  405,  Figs.     London  and  Philadelphia,  1892. 

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Wilder  and  Gage.— Anatomical  technology  as  applied  to  the  domestic  cat.  An  introduction  to 
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Wilson.  Edmund  B.,  with  the  cooperation  of  Edward  Learning.— An  atlas  of  fertilization  and 
Karyokinesis.  Columbia  University  Press,  New  York,  1895.  This  atlas  marks  an  era  in  embry- 
ological  study.  It  has  admirable  text  and  diagrams,  but  the  distinguishing  feature  is  the  large, 
number  of  almost  perfect  photo-micrographs. 

Wood,  J.  G. — Common  objects  for  the  microscope.  Pp.  132.  London,  no  date.  Upwards  of  400 
figures  of  pretty  objects  for  the  microscope,  also  brief  descriptions  and  directions  for  preparation  . 

Wormly,  T.  (',.—  The  micro-chemistry  of  poisons.     2d  ed.     Pp.  742,  illus.     Philadelphia,  1885. 

Wythe,  J.  H. — The  microscopist  ;  a  manual  of  microscopy  and  a  compendium  of  microscopical 
science.     4th  ed.     Pp.  434,  252  Fig.     Philadelphia,  1880. 

Zeiss.  R. — Special-catalog  iiber  Apparate  fiir  Mikro-photographie.  Jena.  Instructions  for 
using  the  apochromatic  objecthes  and  projection  oculars,  etc. 

Zimmermann,  Dr.  A. — Das  Mikroskop,   ein   Leitfaden  der  wissenschaftlichen   Mikroskopie. 
Illustrated.     Leipzig  und  Wein,  1895. 
See  also  Watt's  chemical  dictionary,  and  the  various  general  and  technical  encyclopedias. 

PERIODICALS.* 

The  American  journal  of  microscopy  and  popular  science.     Illustrated.     New  York,  1876-1881. 

The  American  monthly  microscopical  journal.     Illustrated,     Washington,  D.  C,  1880  +  . 

American  naturalist.     Illustrated.     Salem  and  Philadelphia,  1867  f. 

American  quarterly  microscopial  journal,  containing  the  transactions  of  the  New  York  micro- 
scopial  society.     Illustrated.     New  York,   1878. 

American  microscopical  society,  Proceedings.     1878  +  . 

Anatomischer  Anzeiger.  Centralblatt  fiir  die  gesammte  wissenschaftliche  Anatomic  Amt- 
liches  Organ  der  anatomischen  Gesellschaft.  Herausgegeben  von  Dr.  Karl  Bardeleben.  Jena, 
1886  -.  Besides  articles  relating  to  the  microscope  or  histology,  a  full  record  of  current  anatomi- 
cal literature  is  given. 

Annates  de  la  societe.  beige  de  microscopic     Bruxelles,  1S74 

Archiv  fiir  microscopische  Anatomic     Illustrated.     Bonn,  1865+. 

Centralblatt  fiir  Physiologie.  Unter  Mitwirkung  der  physiologischen  Gesellschaft  zu  Berlin 
Herausgegeben  von  s.  Exnerund  J.  Gad.  Leipzig  und  Wien.  1887  h  Brief  extracts  of  papers 
having  a  physiological  bearing.     Full  bibliography  of  current  literature. 

English  mechanic.  London,  1S66  h  Contains  many  of  the  papers  of  Mr.  Nelson  on  lighting, 
photo-micrography,  etc. 

Index  Medicus.     New  York,  1879  Bibliography,  including  histology  and  microscopy. 

International  journal  of  microscopy  and  popular  science.     London,  1890+. 
Journal  of  anatomy  and  physiology.     Illustrated.     London  and  Cambridge,  1S67+. 
Journal  de  micrographic     Illustrated.     Paris,  1877-1892. 
Journal  of  microscopy  and  natural  science.    London,  1885  +  . 


♦Note.— When  a  periodical  is  no  longer  published,  the  dates  of  the  first  and  last  volumes  are 
given  ;  but  if  still  being  published,  the  date  of  the  first  volume  is  followed  by  a  plus  sign. 

See  Vol.  XVI  of  the  Index  Catalog  of  the  Library  of  the  Surgeon  General's  office  for  a  full  list 
of  periodicals. 


BIBLIOGRAPHY. 

Journal  of  the  New  York  microscopical  Illustrated.     New  York 

Journal  of  physiology.    Illustrated     London  and  (.'ami'!.'.' 
Journal  i ii  the  Am.  i  i.  m  chi  mical  • 

journal  ol  th<  ch<  mical  sc*  i<  i>      London,  •.    . 

journal  of  the  royal   microscopical  society,    Illustrated.    Londoi 
works  and  papers  relating  to  the  mici  pical  mi  *  1 1 « >■  1  -~  and  hist 

cludes  a  summary  of  many  "I  the  papers. 

Journal  of  the  Queckett  microscopical  club      London 

The  Lens,  a  quarterly  journal  "i   microscopy,  and  the  allied  natural 
sactions  of  the  state  microscopical  societj  ol  Illinois.     Illustrated.    Chics 

The  Microscope.     Illustrated.     Washington,  D.  C,  1881 
Microscopical  bulletin  and  scienct  news,    Illustrated,     Philadelphia,   i 
ward  Pennock  introduced  the  term  "  par-focal  "  for  oculars  (see  vol.  iii   p 
Monthly  microscopical  journal.     Illustrated.     London.   [869-1877. 
Nature.     Illustrated.     London,  1869+. 
The  Observer.     Portland,  Conn.,  189c 

Philosophical  Transactions  of  the  Royal  .Society  of  London.     Illustrated.     Lond 
Proceedings  of  the  American  microscopical  society,  18; 
Proceedings  of  the  Royal  Society.     London,  1854    , 
Quarterly  journal  of  microscopical  science.    Illustrated.    London 
Science  Record.     Boston,  1883-4. 
Zeitschrift  fur  Instrumentenkunde.    Berlin,  1881 
Zeitschrift  fiir  physiologische  Cliemie.    Strassburg,  1877     . 

Zeitschrift  fiir  wissenschaftliche   Mikroskpie  und   fiir  mikroskoi)ische  Technik.     Illu^t- 
Braunschweig.     1SS4  -  .     Methods,  bibliography  and  original  papers. 

Besides   the  above-named  periodicals,   articles  on  the  microscope   or  the  application  of  the 
microscope  appear  occasionally  in  nearly  all  of    the  scientific    journals.      One  is  likely  to  get 
references  to  these  articles  through   the  Jour.  Roy.  Micr.  Soc.  or  the  Zeit.  wisfl.  Mikrosk<  • 
Excellent  articles  on  Photo-micrography  occur  in  the  special  Journals  and  Annuals  of 
raphy. 


I  X  DEX 


Ahhe  apertometer,  212. 
Abbe  camera  lucida,    1 1  1 

of,  1 1  j  ;  drawing  w  ith 


arrangement 
115-118;  fig- 


ures, 98,  no,  114;  binge  for  prism, 


An  astigmatic  objecti 
Angle  of  aperture,  t6, 
Angstrom  and  Stokes'  law  of  :<: 

spectra,  [23. 
Angular  aperture,  [6,  17. 
Anisotropic,  137. 


117;  inclined  microscope  with,  115      Apertometer,  Al. lie's,  212. 


Abbe  condenser  or  illuminator,  l 

amount  ol  lightfor,43;  dark-ground 
illumination  with,  48;  experiments, 
46;  laboratory  microscope  with,  61  ; 
light,  axial  and  oblique,  46;  mirror 
with,  46. 

Abbe's  test-plate,  method  of  using,  210. 

Aberration,  chromatic,  4  ;  by  cover-glass, 
51  ;  negative,  52  ;  spherical,  4,  210. 

Absorption  spectra  122,  135;  amount  of 
material  necessary  and  its  proper  ma- 
nipulation, [30;  Angstrom  &  Stokes' 
law  of,  1  23  ;  handed,  not  given  by  all 
colored  ohjects,  133  ;  of  blood,  131 


Aperture  ol  objective,  17  22,  212;  angu- 
lar,  10,   17  ;  formula  for,   17,   r8 ;  of 
illuminating  cone,   4  1  ;    num<  rical 
17,  20,  212  ;  numerical  of  conden 
43;  and  optical  section,  S7  ;  signifi- 
cance of,  20. 

Aplauatic  cone,  44  ;  objectives,  12  ;  ocu- 
lar, 25. 

Apochromatic  condenser,  40  ;  objective-. 
[2,  6l,   105. 

Apparatus  and  material,  1,  33,  So,  92,  109, 
i-?".  1  i",  [83  ;  lor  drawing,  216;  for 
photo-micrography,  [85,  [91. 

Appearances,  interpretation,  So,  91. 


of  colored  hodies,  123,  133  ;  of  color-    Aristotype  paper,  20S. 


less  bodies,    134  ;  of  minerals,    13. 
of  permanganate  of  potash,  131. 

Acetylene  light,  36,  48. 

Achromatic  condenser,  40,  41  ;  object- 
ives, 12,  61  ;  oculars,  23  ;  triplet,  7. 

Achromatism,  13. 

Actinic  focus,  [89;  image,  184. 

Adjustable  objectives,  12,  13,52;  experi- 
ments, 51  ;  and  micrometry,  106  ; 
and  photo- micrography,  201. 

Adjusting  collar,  graduation  of,  52. 

Adjustment,  of  analyzer,  136  ;  coarse  or 
rapid,   60;    fine,   60;    focusing,    60; 


Arrangement  of  condenser,  45  ;  of  lamp, 
hull's  eye  and  microscope,  49  ;  mi- 
nute ohjects,  [80 ;  serial  sections. 
169;  tissue  for  sections,  168. 

Artifacts,  Si . 

Artificial  illumination,  36,  46,  4S  ;  for 
photo-micrography,  200. 

Autograph,  photo-micrographic  camera, 
190-192. 

Avoidance  of  diffusion  currents,  162  ;  of 
distortion,  1 1 1. 


Axial  light,  35, 84 ;  experiments,  39 ;  with 
Abbe  illuminator,  46. 

of'objective,  12,  13,  51  ;  of  objective    Axial  point,  16  ;  ray,  35, 

for   cover  glass,  specific    directions.    Axis,  optic,  2,3,  11  ;  of  illuminator,    . 

53  ;  testing,  60  ;  with  graduated  col-  secondary,  5,  47. 

lar,  53. 
Aerial  image,  30,  31.  B 

Air  bubbles,    S 4,    S3  ;     with   central   and 

oblique  illumination,  84,  85  ;  air  and    Back  combination  or  system  of  objective, 


oil,  distinguished  optically,  S5  ;  hv 
reflected  light,  85. 

Albumen  fixative,  Mayer's,  175. 

Albuminous  material,  removal  of,  58. 

Alcohol,  ethylic,  175. 

Alcoholic  dye,  staining  sections  with,  16". 

Alum  solution,  175. 

Amici  prism,  1  20,  126. 

Amplifier,  97,  214. 

Amplification  of  microscope,  92. 

Analyzer,  126,  130;  adjustment  ami  put- 
ting in  position,  1 


IO    1  ;. 
Bacillus  tuberculosis,  56. 

Balsam.  175  ;  bottle,  i'>4  ;  balsam,  mount 

ingin,  163,  167;  preparation  of,  1; 

removal    from   lenses,  58 .    natural, 

170;    neutral,    170;    removal    from 

slides,  141;  x\  lene,  1 75,  1 
Banded  absorption  spectra  not  given  by 

all  colored  ob 
Bands,  absoi ption,  1 
Base,  circular,  >'t  m 

Bed,  earner. 1. 


228 


INDEX. 


Benzin,  removing  of  from  sections,  166. 

B  axial  crystals,  [38. 

Bibliography,  32/91,  108,  119,  135,  139, 
1;;.  iv.  209,  215. 

Binocular  vision,  for  getting  magnifica- 
tion, 93. 

Blood,  absorption  spectrum  of,  131  ;  or 
other  albuminous  materi.nl,  removal, 
58  ;  velocity  of  current,  88 

Body  of  microscope,  Frontispiece. 

Bottle  for  balsam,  glycerin  or  shellac,  164. 

Bowl,  waste,  162. 

Bread  crumbs,  examination  of,  90. 

Bromide  paper,  208. 

Brownian  movement,  88. 

Brunswick  black,  removal  from  lenses. 58. 

Bubble,  air,  84-85. 

Bull's-eye,  49,  191,  203  ;  engraving,  glass 
for,  198. 

Burning  point,  6  ;  finding  of,  30. 

Butterfly  scales,  90. 


Cabinet  for  microscopical  preparations, 

i73>  174- 
Calipers,  micrometer,   143,  144;   pocket, 

143- 
Camera  bed,  207  ;  photographic,  for  draw- 
ing, 208 ;    long   and   short   bellows, 

187,  200;    photo-micrographic,    186, 

188,  190-193,  202.   205,  207  ;  testing, 
1S5  ;  vertical,  18S,  293,  202,  205,  207. 

Camera  lucida,  Abbe,  III  ;  arrangement 
of,  112,  113;  drawing  with,  114,  115, 
118  ;  hinge  for  prism,  112  ;  with  in- 
clined microscope,  115;  laboratory 
microscope  with,  61  ;  magnification 
of  microscope  with,  92,  118. 

Camera  lucida,  definition,  109  ;  figures 
of,  98,  no  in,  1 14  ;  Wollaston's,  95, 
in. 

Canada  balsam,  175  ;  mounting  in,  163, 
167  ;  preparation  of,  175  ;  removal 
from  lenses,  58;  removal  from  slides, 
141. 

Carbol-xylene,  176. 

Carbon-monoxide  hemaglobin,  spectrum 

of,  133- 

Carbonate  of  lime,  pedesis,  89. 

Card  catalog,  173  ;  centering,  149. 

Care  of,  eyes,  58  ;  microscope,  mechan- 
ical parts,  57  ;  optical  parts,  57  ;  neg- 
atives. 197  ;  water  immersion  object- 
ives, 55. 

Carmine  to  show  currents  and  pedesis, 
89  ;  spectrum  of,  133. 

Castor- xylene  clarifier,  176. 

Catalog,  cards,  labels,  ink  for,  173. 

Cataloging,  formula,  172  ;  preparations, 
171-172. 

Celloidin  for  coating  glass  rod,  87. 


Cells,  deep,  thin,  14S  ;  isolated,  prepara- 
tion of,  155  ;  mounting,  148  ;  stain- 
ing. 155- 

Cement,  shellac,  179. 

Cementing  collodion,  177. 

Center,  optical,  2,  3. 

Centering  and  arrangement  of  illumina- 
tor, 41,45;  card,  149;  condenser  for 
])  h  o  t  o  -  m  i  c  r  o  g  r  a  p  hy  ,  201  ;  dia- 
phragm, 42  ;  image  ot  source  of  illu- 
mination, 42 

Central  light,  35,  84;  with  a  mirror,  39. 

Chain  lens  holder,  199 

Chamber,  moist,  151. 

Chimney,  metal  for  lamp,  48 

Chloroform,  paraffin,  176  ;  infiltrating 
with,  165  ;  saturating  with,  165. 

Chromatic  aberration,  4  ;  correction,  12, 
13  ;  correction,  test  tor,  210. 

Chemical  focus,  13  ;  rays,  13. 

Clarifier,  176;  castor-x\  lene,  176. 

Cleaning  back  lens  of  objective,  58  ;  ho- 
mogeneous objectives,  56  ;  mixtures 
for  glass,  145  ;  slides  and  cover- 
glasses,  140-142  ;  water  immersion 
objectives,  55. 

Clearer,  clearing,  153,  164,  167,  176. 

Clearing  mixture,  preparation  ot,  176. 

Clinical  microscope,  76. 

Clothes  moth,  examination  of  scales,  90. 

Cloudiness  of  objective  and  ocular,  how 
to  determine,  82  ;  removal,  58. 

Coarse  adjustment  of  microscope,  Front- 
ispiece ;  testing.  60. 

Cob-web  micrometer,  104. 

Collective,  24 

Collodion,  176;  for  coating  glass  rod,  S7  ; 
cementing,  177  ;  clarifying,  158;  cot- 
ton, 176;  hardening,  158;  method, 
157,  164;  thick,  158,  177  ;  thin,  157, 
177. 

Color  correction,  13  ;  images,  51,  56  ;  law 
of,  124;  production  of,  138;  screens, 

I91.  5o. 
I  Colored  minerals,  absorption  spectra  of, 
134  ;  substances,  spectra  of,  133. 

Colorless  bodies,  spectra  of,  134. 

Coma,  53. 

Combination  of  lenses,  back  and  front, 
10-12,  52  ;  optical,  105. 

Comparison  prism,  127,  128;  spectrum, 
128. 

Compensating  ocular,  24-26. 

Complementary  spectra,  124. 

Compound  microscope,  see  under  micro- 
scope. 

Concave  lenses,  3  ;  mirror,  use  of,  36. 

Condenser,  39-49  ;  Abbe,  44-4S  ;  achro- 
matic, 40,  191,  201  ;  apochromatic,  40, 
191  ;  bull's-eye,  49,  198,  200;  center- 
ing, 41,  45  ;  illuminating  cone  with, 
44  ;  mirror  for,  46  ;  non-achromatic, 


INDE  Y 


l ;  ;   autnei  i<  al   api  rture  of,    i     1 1  ; 
optic  \i,  45  ;  for  photo  mi- 

crograph] ,  40, 191,  201 ;  substage, 
\o     See  ilso  illuminator. 

Condensing  lens,  35. 

Cone,  aplaualic,  1 1  ;  illuminating,  43. 

Conjugate  foci,  3. 

Construction  of  images,  geometrical,  5. 

Continuous  spectium,  123. 
oured,  doubly,  87. 

Converging  lens,  3,  5  ;  lens-system,   10. 

Convex  lenses,  3,  5. 

Corn  starch,  ex  imiuation  of,  91 1 

Correction,  chromatic,  or  color,  13,  210; 
cover-glass,  15,  52-53  ;  cover,  tube- 
length  lor,  53  54;  over  and  under, 

i.v' 

Cost  ol  microscope,  63. 

Cotton,  collodion,  17');  examination  of, 
90  ;  gun,  176;  soluble,  176. 

Cover- glass,  or  covering  glass,  i|i  ;  ab- 
erration by,  J  1  ;  adjustment,  spe- 
cific directions,  53  ;  adjustment  lor, 
in  pho  o  micrography,  201  ;  adjust- 
ment and  tube-length,  [3,  14,  53; 
anchoring,  150;  cleaning,  141,  [42; 
correction,  52,  53;  efftct  on  rays 
from  object,  32;  gauges,  143-145: 
larger  than  object,  146;  measurer, 
[43-145;  measuring  thickness  of. 
143  ;  non  adjustable  objectives  table 
of  thickness  15;  N'o  1,  variation  of 
thickness.  143  ;  putting  on.  84,  146  ; 
sealing,  1  (.8,  150;  size  of,  146;  thick- 
ness "i,  1  |,  15,  143,  170;  tube-length 
with,  14,  53,  54;  wiping,  142;  with 
serial  sections,  170. 

Crayons,  216. 

Crystals,  biaxial,  depolarizing,  13S;  from 
fro^r  for  pedesis,  S9. 

Crystallization  under  microscope,  [8  1. 

Crystallography,  48,  1 80 ;  list  of  substan- 
■  es  fi  >r,  [8 1 

Currents,  diffusion,  avoidance  of,  162  ; 
in  liquids,  88. 

Cutting  sections,  160,  166. 


Dammar,    for    fixing   crayon    drawings, 

;  removal  from  lenses,  58 
Dark  ground    illumination,    36,    47,  4s; 

with    Abbe    illuminator,    \8 ;    with 

mirror    |.8 
Daylight,  lighting  with,  34. 
I  defining  power, 
Dehydration,  157,  [62,  [67. 
Demonstration  microscope,  77. 
Depolarizing  cr\ stals,  1 


nation     of    0CU1 

length, 
I  letermiuation  <>i  field  of  mil 
magnification, 

llleiil  in  water,    I  .  orkiug  I 

lance,  38. 

Developers  tor  photo  ni 
Development  ol  negatii  e,  1 
Diagrams  with  photographic  object) 
reparatii  >u  of,  .'15. 

Diamond,  writing, 

Diaphragms  and  their  en  ploymenf 
I  I  ;   central   sic,;,,      6,     ;:      •  •■     ei 
42,  1"^ ;  iris,  4  1  ;  m  ular,  28  :  pin- hi 
41,  is.:  size  and  position  of  opening, 
14;  with  condenser,  44. 

Diffraction,    grating,    122;    illusory    aji 
pearaui  1  s  due  to, 

1  liffubion  currents,  avoidance  of,  K 

Direct,    light,    34;    vision    sped 
1  21  >. 

I  h'spersing  ])rism,  122. 

Displacements,  in   mounting  objects  in 
1  esinous  media,  [53, 

I  lissecting  mien  >s<  ope,  9. 

Dissociator,    formaldehyde,    177;    nittic 
acid,   179. 

Distance,  principal  1  iidard 

at  which   the  virtual  image  is  mi 
ured,  97  ;  working  d.   of  simple  mi- 
croscope or  objective,  38;  working 
d.  of  compound  microscope,  11,  v,. 

38. 
Distinctness  of  outline,  85. 

.:  tion  iii  drawing,  avoidance  of,  in. 
Diverging  U  its,  3. 
Dividers,  measuring  spread  of,  93. 
Double  sp<  ctrum,  12s  ;  vision,  92,  9 
Doubly  contoured,  S7  ;  refracting, 
Draw- ml  ie,  Frontispitce ;  pushing  it 
Drawing,  with  Abbe  camera  lurida,  114 

its;  hoard  for  Abbe  camera  lucida, 

115    [  t6;  distortion  avoidant  e  of,  III; 
embryograph  for,  119;  with  mi.  1 
scope,     109 ;    photographic    can 
for,   iim;  for  photo  engravinj 
scale  and  enlargement  of,  1  18  ;  Iran 
ferring,  2 1  7. 

Dry  objectives,  11,  17   10 ;  fo 

microscope,   m  ;   light  utili/<  d,    1 
dry  mounting,  1  r  ;  numeri<  il  ft] 
ture,    is ;   dry   plates,  di  by 

Maddox,  1 

Drying  neg  itives,  rack  fi 

Dust,  of  living  rooms,  exautn 

:  011  objectives  and  oculars,  1 
to  detei  urn 

Dye,  gem  lining  \*ith  que 

mis,  i6r,  [I 


230 


INDEX. 


Eccentric  diaphragm,  42, 

Etnbryograph,  1  19. 

Engraving  <j,lass  for  bull's  eve  condenser. 

Erect  image,  1. 

Equivalent  focal  length,  11,  214  ;  focus  of 
objectives.  11,  26,  214  ;  focus  of  ocu- 
lar, 26,   214. 

Elements,  histological,  isolation  of,  154. 

Eosiu,  1  77. 

Ether,  sulphuric,  177. 

Ether- alcohol,  177;  saturated  with,  157 

Ethylic  alcohol,  175. 

Ex  imination  of  (lust   of  living   rooms, 
bread  crumbs,  corn  starch,   fibres  of 
cotton,  linen,  silk,  human   and   ani- 
mal hairs,  potato,  rice,  scales  of  but 
terflies  and  moths,  wheat. 

Experiments,  Abbe  illuminator,  46;  with 
adjustableand  immersion  objectives, 
51  ;  compound  microscope,  26;  crys- 
t  dlography  and  micro-chemistry, 
180;  homogeneous  immersion  ob- 
jective, 55  ;  lighting  and  focusing, 
;  in  micro  chemistry,  180  ;  with 
micro  spectroscope,  131  ;  with  micro- 
poliriscope,  137  ;  in  mounting,  146- 
161  ;  photo-micrography,  193;  simple 
microscope,  6. 

Exposure  of  photographic  plates,  194, 
199,  200,  203. 

Extraordinary  ray  of  polarize  1  light,  136 

Eye  and  microscope,    1,  6,  10,  31. 

Eves,  careof.  5S  ;  mi-c  e  volitantes  of,  90 

Eye-lens  of  the  ocular,  22. 

Eyepiece,  22  ;  microme;er;  102. 

Eye-point,  7,  22,  no;  of  ocular,  demon- 
stration, 32. 

Eye-shade,  Ward's,  59  ;  double,  59. 


Farrant's  solution,  in  mounting  objects, 
151  ;  preparation  of,  177. 

Feather,  examination  of,  90. 

Fibers,  examination  of,  90 

Field,  27;  with  camera  lucida,  95  ;  illu 
ruination  of,  44.  49  ;  with  orthoscopic 
ocular,    23  ;  with    periscopie   ocular, 
24  ;  of  view  with  microscope,  27.  29, 
93,  109;  size  of,  with  different  object 
ives  and  oculars,  27,  29 

Field  lens,  of  ocular,  22  ;  action  of,  31  ; 
dust  on,  «S2. 

Filar  micrometer  ocular,  23  ;  ocular  mi- 
crometer, 103. 

Filter  paper,  Japanese,  57. 

Filters,  light,  191. 

Filtering  balsam,  etc.,  paper  funnel  for, 

175- 


Fine  adjustment,  Frontispiece  ;  testing, 
60 

Fir,  balsam  of,   1  75. 

Fixative,  albumen,  Mayer's,  175. 

Fixing,  reagents  for,   175  ;   tissue,   157. 

Flame,  image  of,  43 

Fluid,  immersion,  55,56;  testing.  213. 

Focal  distance,  or  point,  principal,  30; 
length,  equivalent,   11. 

Focus,    6;   always   up,    ^8;  actinic,    1S9  ; 
chemical,    13  ;   conjugate,   3  ;  of  ob- 
jectives,  equivalent,    II,   26,   214  ;  of 
oculars,  equivalent,  26,214  ;   optical, 
15  ;  principal,  3.  5  ;    visual,  1S9 

Focusing,  6,  33  ;  adjustments,  testing,  60; 
with  compound  microscope,  33  ;  ex- 
periments, 36  ;  glass.  197-19S  ;  with 
high  objectives.  37  ;  with  low  object- 
ive>,  36  ;  objective  for  micro  spectro- 
scope,  130  ;  for  photo  micrography, 
194,  199,  201  ;  screen  for  photo  mi- 
crography, 194-195;  with  simple  mi- 
croscope, 31  ;  slit  of  micro  spectro- 
scope. 131. 

Form  of  objects,  determination  of,  83. 

Formal,  177. 

Formaldehyde  dissociator,  177  ;  for  iso- 
lation, 154. 

Formula  for  cataloging,  172. 

Fraunhofer  lines,  123. 

Front  combination  or  lens  of  objective, 
10.  1 1. 

Frontal  sections,  170. 

Function  of  objective,  29  30;  of  ocular, 

30. 
Funnel,  paper,  175. 


G 


Gauge,  cover-glass,  143,  145. 

Gelatin,  liquid,  preparation  of,  17S. 

Geometrical  construction  of  images,  5. 

Glass,  cleaning  mixture  for,  145  ;  focus- 
big,  '97-198;  ground,  29;  for  object- 
ives, 12,  63  ;  rod  appearance  under 
microscope,  86,  87  ;  slides  or  slips, 
141 1. 

Glue,  liquid,  preparation  of,  178. 

Glycerin,  bottle  for,  164;  mounting  ob- 
jects in.  order  of  procedure,  149,  177; 
removal.  58. 

Glycerin  jelly,  mounting  objects  in,  or- 
der of  procedure.  150;  preparation 
of,   177 

Gold  size,  removal  from  lenses,  5S. 

Goniometer  ocular,  23. 

Graduation  of  adjusting  collar,  53. 

Grating,  diffraction,  122. 

Ground  glass,  preparation  of,  29. 

Gun  cotton,  176. 


INDEX. 


'3i 


H 


Hairs,  examination  of,  90. 
Half-tones  from  photo  micrographs,  2 

216. 
Hardening   collodion,   [58;    tissue,   15,7, 

[64. 
Heliostat,  209. 
Hematoxylin,  17s. 
I  [emoglobin  sptectrum,  1 
Herapath's  method  of  determining  mi- 

nute  quantities  of  quinine,  [81. 
Histological  elements,  isolation  of,  154. 
Histology,  physiological,  [73. 
History  of  photo-micrography,  ;  s  j . 
Holder,  lens,  !S  ;  needle,  146. 
Homogeneous  innnersion  objective,  17- 

[9;  cleaning,  56;  experiments,  55  ; 

for  laboratory  microscope,  61  ;  light 

utilized,  iS,  21  ;  numerical  aperture, 

21. 
Homogeneous  liquid,  12,  213  ;  tester  for, 

55.  213. 
Huygeniau  ocular,  22,  24,  31. 

I  "J 

Illuminating,  cone,  aperture  of,  43,  4  |  ; 
for  condenser,  44;  objective,  14; 
power,  21,  22. 

Illumination,  for  Abbe  camera  lucida, 
116;  artificial,  36,  48;  artificial  for 
photo-micrography,  194;  centering 
image  of  source  of,  41-45;  central 
with  air  and  oil,  84,  85  ;  dark-ground, 
36,47,  4S  ;  daylight,  34;  of  entire 
field,  49  ;  lamp  for,  48  ;  metliods  of, 
34,  47;  for  micro-polariscope,  137; 
for  micro-spectroscope.  129  ;  oblique, 
with  air  and  oil,  84,  S5  ;  for  photo- 
micrography, 192  ;  for  Wollaston's 
camera  lucida,   1  11 . 

Illuminator,  39-40  ;  Abbe,  j  |,  \~  \  Abbe, 
axial  and  oblique  ligbt,  36  ;  Abbe, 
experiments,  46;  Abbe,  mirror  and 
light  for,  46;  achromatic,  40;  cen- 
tering and  arrangement,  45  ;  immer- 
sion, 45.     See  also  condenser. 

[mage,  actinic,  [84;  aerial,  30,  31  ;  cen- 
tering i.  of  source  <>f  illumination, 
42;  color,  49,  51.  56  ;  with  dry  apo- 
ehromatic  and  water  immersion  ob 
jectives,  13;  erect,  I  ;  inverted,  7; 
inverted,  real  of  objective,  29  :  of 
flame,  43  ;  formation  of.  3;  geomet- 
rical construction  of.  5  ;  and  obji 
size  and  position,  5,  11,  96;  real,  5, 

■  •.  29   ;i ,  92  ;  refraction,  | 
retinal.  6,    i",  31  ;  swaying  of.     |<>  ; 
virtual  i.  and  standard   distance  at 
which  measured,  <>,  31,  92. 

[mage-power  of  objectives,  1  <». 


Embedding,  158, 

Immersion    fluid,    55,    ?  1  \  .    illumin. 
;  liquid,  55 

Incandescence  or  line  bj 

Incident  light, 

index  ..1  (,f  medium  in 

front  of  objective,  [7-18. 
Infiltration  with  chloroform,    U 

lodion,  137,  [58;  ether,  157  ;  paraffin, 

[65. 
Initial  magnification,  214. 
Ink    for   labels   and  .    for 

drawing,  216. 
Interpretation  of  appearances  under  the 

microscope, 
Inverted  image,  7,  29. 
Iris  diaphragm,  40,  181. 
Irrigating  with  reagents.   1 
[sochromatic  plates,  [94,  rg 
Isolation,  154  ;  with  formaldehyde,  131 

nitric  acid,  155. 
Isotropic,  137. 

Japanese  filter  or  tissue  paper,  57,  160. 
Jelly,  glycerin,  177. 
Jena  glass,  63. 
Jurisprudence,  micrometry  in,  108. 


Labels  and  catalogs,  ink    for    175;   | 

aration  of,  1 70. 
Labeling      microscopical     preparal 

171  ;    photographic    negative,    [< 

serial  sections     171. 

Laboratory  compound  microscope,  61. 
Lamp,  microscope,  18;  spirit,  t< 
Lamp-light,  36. 
Law  Of  color,   1  24. 
I. ens,  concave,  5  ; 

vex.  3  ;  ey< 

8,  155,  156, 

ro. 
Lens  systems,  10. 
Letters  in   stairs.   82  ;    for  photo-eugrav- 

ing,  2l8, 
Lettering  oculars, 
Light,  with   Abbe   illuminator. 

t\  leue,   36,    |.8  ;  artificial, 

35,  59;  axial  with  Abbe  illumina! 

[6  ;  direct,  3 1  ;  central,  | 
.1  ;   incident.    ;  1  ;    with   mim  I 
oblique  with  Al 

illuminatoi 

to  micrograph) ,  1 

reflected  incident  ot  di 

mitted,  35  ;  utilized  with  .1 
length  ■ 
Lighting,      ;      !-•     Vbl         tmera  lu 

115;  artificial,  ; 

and     focusing 

polarisco] 


converging,    5  ; 
field,  22,  26  ;  holder. 
paper,  57  :  system, 


232 


IXDEX. 


scope,  129;  with  a  mirror,  36  ;  with 

daylight,  34;  for  photo-micrography, 

192. 
Line  spectrum,  1  23. 
Linen,  examination  of,  90. 
Liquid,   currents  in,  88;   homogeneous, 

55i   213. 


M 


Macro-photography,  1 8  ]. 

uification  of  compensating  oculars, 
in  ;  effect  of  adjusting  objective,  io5  ; 
determination  of,  92,  94 ;  expressed 
in  diameters,  92  ;  initial  or  indepen- 
dent, 214;  method  of  binocular  or 
double  vision  in  obtaining,  93  ; 
of  microscope,  92  ;  of  microscope 
with  Abbe  camera  lucida,  118;  of 
microscope,  compound,  94  ;  of  micro- 
scope, simple,  93  ;  of  photo-micro- 
graphs, determination  of,  202  ;  real 
images,  92  ;  table  of,  with  ocular  mi- 
crometer, 99 ;  varying  with  com- 
pound microscope,  97  ;  and  velocitv, 
88. 

Magnifier,  tripod,  7,  198. 

Marker  for  preparations,  63,  64. 

Marking  objects,  63,  64,  94,  101  ;  nega- 
tives, 197  ;  objectives,  65. 

Material  and  apparatus,  1,  33,  80,  92,  120, 
140,  175-181,  183,  210,  216. 

Measure,  unit  of,  in  micrometry,  100  ;  of 
wave  length,  128. 

Measurer,  cover-glass,  143-145. 

Measuring  the  spread  of  dividers,  93  ; 
thickness  of  cover-glass,  143. 

Mechanical  parts  of  compound  micro 
scope,  61  ;  of  microscope,  care  of, 
57  ;  testing,  60 

Mechanical  stage,  61,  65-66. 

Medium,  mounting,  147. 

Met-hemaglobiu,  spectrum  of,    122,   133. 

Methods,  collodion,  157  ;  paraffin,  164. 

Micro-chemistry,  180. 

Micrometer,  92  ;  calipers,  143,  144  ;  cob- 
web, 104;  filar  in.  ocular,  103;  filling 
lines  of,  94  ;  net,  r  13  ;  lines,  arrange- 
ment of  ocular  and  stage,  107  ;  object 
or  objective,  94  ;  ocular  or  eye-piece, 
102,  103  ;  ocular,  micrometry  with, 
105  ;  ocular,  ratio,  106  ;  ocular,  valu- 
ation of,  103  ;  ocular,  varying  valua- 
tion of,  105  ;  screw  ocular,  103  ;  stage, 
94  ;  table  of  magnification,  99. 

Micrometry,  definition,  100-102  ;  with 
adjustable  objectives,  106  ;  compari- 
son of  methods,  107  ;  with  compound 
microscope,  100;  and  jurisprudence, 
10S  ;  limit  of  accuracy  in,  107;  with 
ocular  micrometer,  105  ;  with  simple 


microscope,  100;  remarks  on,  106; 
unit  of  measure  in,  100. 

Micro-millimeter,  101. 

Micron,  100;  for  measuring  wave-length 
of  light,  129. 

Micro-photograph,  183. 

Micro-photography,  distinguished  from 
photo- micrography,  183. 

Micro-polariscope,  89,  136;  experiments, 
137  ;  for  laboratory  microscope, 
lighting  for,  137  ;  objectives  to  use 
with,  136  ;  purpose  of,  137  ;  selenite 
plate    with,    138  ;    sulphonal    with, 

139- 

Micro-polarizer,  136. 

Microscope,  definition,  1  ;  amplification 
of,  92;  clinical,  76;  demonstration, 
77  ;  dissecting,  9  ;  care  of,  57  ;  eye 
and,  1,6,  10  ;  field  of,  27,  29  ;  focus- 
ing, 31  ;  magnification,  92  ;  (or  photo- 
micrography, 187;  polarizing,  136; 
price  of,  61,  63  ;  putting  an  object 
under,  27  ;  screen,  56. 

Microscope  compound,  definition,  7  ; 
drawing  with,  110;  figures,  10,  65; 
focusing,  36-37  ;  for  laboratory,  61  ; 
lamp,  48  ;  magnification  or  magnify- 
ing power,  94  ;  magnification  and 
size  of  drawing  with  Abbe  camera 
lucida,  118  ;  mechanical  parts  of,  61  ; 
micrometry  with,  100  ;  optic  axis  of, 
10;  optical  parts  of,  10,61  ;  polariz- 
ing, pedesis  with,  89  ;  varying  magni- 
fication, 97  ;  working  distance  of,  38  ; 
testing,  60. 

Microscope,  simple,  definition,  1  ;  exper- 
iments with,  6  ;  figures,  6-8,  31,  155- 
156,  197-199;  focusing  with,  33; 
magnification  of,  93  ;  micrometry 
with,  100  ;  working  distance  of,  33. 

Microscopic  objective,  10  ;  objective  low, 
attached  to  camera,  198  ;  objects, 
drawing,  109  ;  ocular,  22  ;  slides  or 
slips,  140. 

Microscopical  preparations,  cabinet  for, 
174;  cataloging,  172;  labeling,  171; 
mounting,  146-170;  tube-length,  14, 

15- 

Microscopy  and  photography,  183. 

Micro-spectroscope,  120-121  ;  adjusting, 
125  ;  experiments,  131  ;  focusing, 
130  ;  focusing  the  slit,  125  ;  for  labo- 
ratory microscope,  61  ;  lighting  for, 
129  ;  objectives  to  use  with,  130  ;  re- 
versal, apparent,  of  colors  in,  120  ; 
slit,  mechanism  of,  121,  125. 

Micrum,  100. 

Mikron,  100. 

Minerals,  colored,  absorption  spectra  of, 

134- 
Minute  objects,  arrangement  of,  180. 


INDEX. 


Minor,  i<>,  1 1  ;  for  Abbe  illuminator,  i'>. 
of  camera  lucida,  arrangement   for 
drawing,   (12;  concave,  use  ofi 
dark-ground  illumination,  17;  li.^bt 
with,  central  and  oblique,  v.i ;  light 
ing  with,  36  ;  plane,  use  of, 

Mixture,  clearing,  1  76. 

Moist  chamber,  151. 

Molecular  movement,  88. 

Monazite  sand,  spectrum  of,  1 

Mono-refringent,  137. 

Mounting,  cells,  preparation  of,  [48  ;  me- 
dia and  preparation  of,  175     79;  ob- 
jects  for  polariscope,    137;   perma 
nent,  147;  temporary    [46 

Mounting  objects,  dry  in  air,  order  of 
procedure,  [47  ;  in  glycerin,  order  of 
procedure,  [49;  in  glycerin  jelly,  or- 
der of  procedure,  150;  in  media  mis 
cible  with  water.  149;  minute  ol> 
jects,  1S0;  permanent,  1(7;  in  resin- 
ous media,  152  ;  in  resinous  media, 
by  'Irving  or  desiccation,  order  of 
procedure,  152  ;  in  resinous  media, 
by  successive  displacements,  order 
of  procedure,  153  ;  temporary,  146. 

Movement,  lirownian,  or  molecular.  88. 

Muscae  volitantes,  90 

Muscular  fibers,  isolation  of,  155. 


N 


Natural  balsam,  170. 

Needle-holder,  146. 

Negative  aberration,  52  ;  development 
of,  196  ;  labeling  and  care  of,  197  ; 
marking,  197  ;  oculars,  22  ;  rack  for 
dr\  iiig, 

Net-micrometer,  113. 

Neutral  balsam,  176. 

Nicol  prism,   136. 

Nitrate  of  uranium,  spectrum  of,  135. 

Nitric  acid,  dissociator.  179. 

Nomenclature  of  objectives,  1  1 

NFou-achromatic  condenser,  44  ;  object- 
ives, 1  2. 

Non-adjustable  objectives,  13  ;  thickness 
of  cover  glass  for,  table,  15. 

Normal  salt  solution,  179. 

Nose-piece,  9,  27  ;  marking  objectives 
on.  65. 

Numerical  aperture  of  condenser,  43  ;  ob- 
jectives, 17,  212;  table  of,  20 ;  reso- 
lution and,  21. 


'  >'    ect,  determination  of  form,  83  ;  hav 
ing  plane  or  irregular  outline-,,  rela- 
tive position  in  a  microscopical  prep 
aration,  ^2  ;  and  image,  size  of,  5,  11, 
96;   marking  parts  of,  63,  64;  mi- 


cromeli  mounting, 

ting  under  mi 

56  ;  suitable  for  photo-mi  hy, 

[91  ;  transparent   with   curved   out- 
lines, :  position  in  uiicri 
preparations, 
objective.  7,  10  ;  achromatii 
adjustable,  1  2,  1 3,  52  :  adj 
perimeuts,  51  ;  adjustable,  niicrom- 
etrj  with,«io6;  adjustable,  photo  mi- 
crograpl  y  with,  201  :  adjustment  for, 
5 1  ;  aerial  image  1  if  anasligni 
[96  ;  aperture  of,   21, 
11  a  tic,    1 2  ;    apocbromatic,    l 
back  combination  of,   12;  cleaning 
back  lens  of,   58  .  1  ollai .   pi  ado 
for  adjustment,    : "-,  .    1  loudii 
dust,  bow  to  determine,  82  ;  d< 
nation  of,  1  1  ;  dry,  11,17  n- 
alent  focus  of,   1 1,  2b,  214  ;  field 

8,   29  ;    focusing    for   mil  I 
scope,  [30;  front  combination  of, 
1  1  ;    function    of,    29,    30  ;   gl 
ti-13,63;  high,  focusing  with,   ■ 
homogeneous  immersion,  17   19;  ho- 
mogeneous immersion,  cleaning, 
homogeneous  immersion,  experi- 
ments, 51,  55  :  illuminating,  14  ;  im- 
age,   power   of,    [9  ;   immersion.    I 
index    of   refraction    of    medium    ill 
front  of,  17.  18;  inverted,  real  ini  • 
of,  29  ;  for  laboratory  microscope, ' 
lettering,  11  ;  li.^bt  utilized  with, 
low,  attached  directlj  tocami  1 
low,   focusing  with,   36  ;    magnifi 
tion  of,   21  1  ;  marking,  by   Krauss' 
method,  6s  ;  to  use  with  micro  polar- 
iscope, [36;  microscopic,  10;  to 
with     micro—:  1 . JO  :     for 

micro  spei  e,     focusing.     1 

nomenclature  of,  ir  :   lion  achromat- 
ic,  12;  non-adjustable,  13;  nou 
justable,  thickness  of  .-over  glass  foi . 
table,    15;   numbering,    11  :   numeri- 
cal aperture,  21,  22.  212  ;  oil  imm 
siou,  12;  panto-chromatic,  1- 
chromatic,  13  ;  perigraphic, 
photo- micrography,  1  on, 

14,  195;  putting  in  position  and 
moving,  26  ;  semi  apochromat  ■ 
table  of  field,  28  ;  termini  logy  of,  u  : 
unadjusiable,    13  :  vai 
ml  and  actini  f,  in  photo-mi- 

crography,   iv  er   immersion, 

17   iw.  55  ;   experimei  irk- 

ing distance  Of,   I  I.  33' 

Oblique  light  unii 

nator,  "th 

a  miii' 

ocular,  22  ;  achromatic 
aplanatic, 
oudiness,  bow  to  determine 


234 


INDEX. 


move,  82  ;  Campani's,  and  cob-web  I 
inicrometer,  23  ;  compensating,  24, 
26  ;  compound,  2;,  ;  continental,  23  ; 
deep,  23  ;  designation  by  magnifica- 
tion or  combined  magnification  and 
equivalent  focus,  26  ;  diaphragm,  28; 
du^t,  how  to  determine,  82  ;  equiva- 
lent focus  of,  26  ;  erecting,  23  ;  eye- 
point  of,  demonstration,  32  ;  field- 
lens,  31  ;  filar  micrometer,  23,  26, 
104  ;  focus,  equivalent  of,  26  ;  func- 
tion of,  3<  1,  3 1  ;  goniometer,  23  ;  high, 
23  ;  holosteric,  23  ;  Huygenian,  22- 
24,  31  ;  iris  diaphragm,  18 1  ;  index, 

23  ;  Jackson  micrometer,  23  ;  Kell- 
ner's,  23  ;  lettering  of,  26  ;  low,  23  ; 
micrometer,  23,  25,  102,  105  ;  mi- 
crometer, micrometry  with,  105  ;  mi- 
crometer ratio,  106;  table  of  magni 
hcation  of,  99  ;  micrometer,  valua- 
tion of,  102  ;  micrometer,  varying 
valuation,  105  ;  micrometer,  ways  of 
using,  105  ;  micrometric,  23  ;  micro- 
scopic, 22,  23  ;  negative,  22,  23  ;  num- 
bering, 26  ;  orthoscopic,  23,  28  ;  par 
focal,  16,  24,  37  ;  periscopic,  24,  28  ; 
for  photo-micrography,  25,  189;  pho- 
to-micrography with  and  without o., 
198,  199;  pointer,  63  ;  positive,  22  ; 
projection,  25,  189  ;  projection,  des- 
ignation of,  189  ;  projection,  use  of 
in  photo- micrography,  199,  202  ;  put- 
ting in  position  and  removing,  27  ; 
Ramsden's,  24  ;  screw  micrometer, 
26,  103  ;   searching,  24,  25  ;  shallow, 

24  ;  solid,  24  ;  spectral,  24,  106  ;  spec- 
troscopic, 24,  120;  stauroscrpic,  24; 
stereoscopic,  24  ;  table  of  field  of,  28  ; 
working,  25. 

Oil,  and  air,  appearances  and  distinguish- 
ing optically,  85  ;  removal,  58  ;  re- 
moval from  sections,  161. 

Oil-globules,  with  central  illumination, 
84  ;  with  oblique  illumination,  85. 

Oil-immersion  objectives,  12. 

Optic  axis,  2,  5  ;  of  condenser,  or  illumi- 
nator, 35  ;  of  microscope,  6. 

Optical  center,  2,  3;  combination.  104, 
105  ;  focus,  13  ;  parts  of  compound 
microscope,  to,  61  ;  parts  of  micro- 
scope, care  of,  and  testing,  57,  60; 
section,  87 

Order  of  procedure  in  mounting  objects, 
dry  or  in  air,  147  ;  in  glycerin,  149  ; 
in  glycerin  jelly,  150;  in  resinous 
media  by  desiccation,  152  ;  in  resin- 
ous media  by  successive  displace- 
ments, 153 

Ordinary  ray  with  polarizer,  136. 

Orthochromatic  plates,  191. 

Orthoscopic  ocular,  field  with,  28. 

Outline,  distinctness  of,  85. 


Over-correction,  5. 

Oxy-hemoglobin,  spectrum  of,  122,  132. 


Pantachromatic  objective,  13. 
Paper,  aristotype,   208  ;  bibulous,  filter, 
lens,  or  Japanese,  57,  160;  bromide, 
208  ;  for  cleaning  oculars  and  object- 
ives,  57,    160;    funnel,    175;   Usago, 
160. 
Parachromatic  objective,  13. 
Paraffin,   179;   chloroform,   176;  chloro- 
form,   infiltrating   with,    165  ;  hard, 
166  ;    imbedding   in,    165  ;    method, 
164  ;    removal   from  lenses,   58  ;  re- 
moving from  sections,  166. 
Parfocal  oculars,  16,  24,  37. 
Parts,  optical  and  mechanical  of  micro- 
scope, 10,  61  ;  testing,  60. 
Pedesis,    SS  ;    compared   with    currents, 
88  ;  with  polarizing  microscope,  89  ; 
proof  of  reality  of,  89. 
Penetrating  power,  21,  22. 
Penetration  of  objective,  21. 
Perigraphic  objective,  196. 
Periscopic  ocular,  field  with,  28. 
Permanent  mounting,  147  ;  preparations 

of  isolated  cells,  155. 
Permanganate  of  potash,  absorption  spec- 
trum of,  122,  131. 
Picric-alcohol,  179. 
Pinhole  diaphragm,  45. 
Pipette,  161. 
Photo  engraving,  216,  219  ;  drawing  for, 

216-219  ;  lettering  for,  218. 
Photographic  negatives,   marking,   197  ; 
objectives,  196  ;  for  photo  microgra- 
phy, 196. 
Photography,  basis  for  figures,  206  ;  com- 
pared with  photo-micrography,  1S3  ; 
indebtedness    to    microscopy,     183, 
184  ;  lighting  large  objects  for,  194  ; 
objectives  for.  196  ;  of  objects  in  alco- 
hol  or  water,    205  ;  with   a   vertical 
camera,  205.  207. 
Photogravures  from  photo-micrographs, 

208. 
;  Photo  micrograph,  183;  developers  for, 
197  ;  determination  of  magnification 
for,  202  ;  at  5-20  diameters,  193  ;  20- 
50  diameters,  198  ;  100-150  diame- 
ters, 201  ;  50  >-2000  diameters,  203  ; 
objects  suitable  for,  191  ;  prints  of. 
20S  ;  plates  for,  194  ;  reproductions 
of.  20S  ;  with  and  without  an  ocular, 
198 
Photo-micrographic  camera,  186,  188, 190, 

192,  193,  202. 
Photo-micrography,  1S3-204;  cover-glass 
correction,  201  ;  apparatus  for,  185  ; 
compared   with  ordinary  photogra- 


/  VDEX. 


phy,   184  ;   condenser  tor,  4  >,    1**1  . 
distinguished  from  micro-photo] 
phy,  [83;  experiments,  193;  exp 
ure  for,   s  03 ;  fo- 

cusiug  i""r,  [95  ;  focusing  screen  for, 
[95  ;  lighting,  [92,  [93;  19 
jectives  and  oculars  lor,  1  1,  189,  1 
vertical  camera  with,  205  ;  visual  and 
aclinic  foci  in,  [89;  with  loiij^  and 
short  bellows,  [87  ;  with  and  without 
ocular,  [89   199;  record  table  for,  204, 

Physiological  histology,  173. 

Plane  minor,  use  of,  36. 

Plate-,   exposure  of,   [93,   [94  >o, 

203  ;  gelati no-bromide,  [84  ;  isochro- 
matic,  or  orthochromatic,   189,   191, 

t97- 
Pleocliroisin,  [38 

Pleurosigma  angulatum,  39 

Point,  axial,  t6  ;   burning,  6. 

Polariscope,  1  27,  136. 

Polarized  light,  extraordinary  and  ordi- 
dinary  rav  of,  136. 

Polarizer  and  analyzer,  putting  in  posi 
tion,  136. 

Polarizing  microscope,  pedesis  with,  S9. 

Position  of  objects  or  parts  of  same  ob- 
ject, 82. 

Positive  oculars,  11,  22. 

Power  of  microscope,  92  ;  illuminating, 
penetrating,  resolving,  21  ;  of  object- 
ive, 19,  215  ;  of  ocular,  26,  215. 

Preparation  of  Canada  balsam.  Farrant's 
solution,  glycerin,  glycerin  jell  v,  etc. 

'  75-179 

Preparation  of  clearing  mixture,  liquid 
gelatin  and  shellac  cement,  [75    179. 

Preparations,  cataloging,  171,  172;  cabi 
net  for,  173    171  ;  labeling,  171  ;  per- 
manent, ot  isolated  cells,    155  ;  stor- 
ing, 171. 

Preparation  of  diagrams,  215  ;  of  ground 
glass,  29  ;  vials,  159. 

Price  of  American  and  foreign  micro- 
scope, 61,  63. 

Principal  focus,  3,  5  ;  focal  distance,  3, 
30  ;   optic  axis,  2,  5. 

Prism  of  Abbe  camera  lucida,  1  1  1  ;   Am- 
ici,   126;  comparison,  127,  12s  ;  <lis 
persing,  [26;  Nicol,  [36;  and  slit  of 
micro  pe,  mut ual  arrange- 

ment,   i2s;    of  Wollaston's  can: 
lucida,  in. 

Prints  and  mechanical  printing  of  photo 
micrographs, 

Projection  objective,   14,   10;,  ;   ocular, 
26,  1 89  ;  desi  fti  il  ion  of,  189  ;  in  pho- 
to micrography,  [9  1, 

Putting  on  cover  i^lass,  1  (.6  ;  an  object 
under  microscope,  27  ;  an  objective 
and  ocular  in  position,  26,  27. 

Pyroxylin,  1  70. 


Q    -R 

1  bant  tor  camera  lucida,  1  ix. 
Quinine,  Eierapath's method  rxnin- 

iiiK  minute  quantit 
Rack  for  drying  negativ< 
Ratio,  ocular  micrometi 

1  75  :  in  with, 

[50  ;  for  mounting,  1 73 
Real  image,  5,  1 

tion,    1 
Record  table  tor  photo-m 
Reflected  light,  34. 

iction,  5<  1 ;  imagi  5  5  ;  inde 

5  >  ;  of  medium  in  front  of  object; 

Refractive,    doubly,     137;    highly, 
singly,  137. 

Relative  position  ot  objects,    Sj. 

Resinous    media,    mounting  objects  in, 
order   of  procedure,    by   drying 
desiccation,  132;  by  a  series  of 
placements,  [53. 

Resolution  and   numerical    aperture,    21. 

Resolving  power,  20. 

Retinal  image,  6,  10. 

Revolver,  27. 

Revolving   nose-piece,    marking    ol 
tives  on,  65. 

Rice,  examination  of,  90. 


Sagittal  section.-,  1  71 1 

Salicylic  acid,  crystallization,   . 

Salt  solution,  normal,    1 

Scale  of  drawing,  n8;  of  wave  lengths, 

128. 
Scales  ofblltterflies  and   moils,  examin- 
ation of,  90. 

.1  ;  focusing  s.  for  pti 

micrography,    194,    :><>  ;   of  ground 

glass,  29  ;  lor  microscope. 
Screw,  society,  62  ;  micromel  104. 

Sealing  cover  glass,  1  p3. 
Searching  ocular,  2 
Se<  ondary  axis,  ; 
Section,  optical, 
Sections,  arrangement  of  tissue  t"<>r. 

clearing,     167  ;    cutting, 

fastening  to  slide, 

170;     removing     benzin,     oil     and 

paraffin  from.  [66,  [61  ;  sagittal,  1 

serial,    168   17 

transfen  in 
Sediment    in    water    determination    ol 

character, 
Selenite  or  polarii 

Semi-apochrout 
Serial  sections, 

in-  171 ;  determining  thickni 


INDEX. 


of,  170  ;  stage  for,  65  ;  thickness  of 
cover- glass  for,  170. 

Shellac  cement,  preparation  of,  179;  re- 
moval from  lenses,  58. 

Sight,  injury  or  improvement  in  micro- 
scopic w>rk,  59. 

Significance  of  aperture,  20. 

Silk,  examination  of,  90. 

Simple  microscope,  see  under  micro- 
scope, I. 

Slides,  1  {<>;  cleaning.  1  \o. 

Slips,    140. 

Slit  mechanism  of  micro-spectroscope, 
12  1. 

Society  screw,  62. 

Sodium,  lines  and  spectrum,    122,  123. 

Solar  spectrum  or  s.  of  sunlight,  1 22. 

Soluble  cotton,  176. 

Solution,  alum,  175  ;  Farrant's,  177. 

Spectral,  colors,  123;  ocular,  120,  126. 

Spectroscope,  direct  vision,  120. 

Spectroscopic  ocular,  24,  120. 

Spectrum,  122  ;  absorption,  123  ;  amount 
of  material  necessary  and  its  proper 
manipulation,  130;  analysis,  135; 
Angstrom  and  Stokes'  law  of,  113; 
banded,  not  given  by  all  colored 
objects,  133  ;  of  blood,  131  ;  of  car- 
bon monoxide  lieinajilobin,  133  ;  of 
carmine  solution,  133  ;  of  colored 
minerals,  134  ;  of  colorless  bodies, 
134;   comparison,  12S;  complemen- 


tary 
12S; 


124  ;  continuous,    123  ;  double, 
incandescence,  123;  line,    123; 


T    -»   I 

J33 


monazite 


met-hemaglobin, 

sand,  134  ;  nitrate  of  uranium,  135  ; 
oxy-hemaglobin,  132;  permanganate 
of  potash,  131  ;  single-handed  of  be- 
maglobin,  132  ;  sodium,  122,  123  ;  so- 
lar, 122,  123  ;  two-banded  of  oxy-he- 
maglobin,   132. 

Spherical  aberration,  4;  test  for,  210. 

Stage,  61  ;    mechanical,  6r,    65,  66  ;  mi- 
crometer, 94  ;  for  serial  sections,  65. 

Stain,  alcoholic,  161  ;  aqueous,  161. 

Staining  cells,  155  ;  sections,    161,  167. 

Stand,  of  microscope,   61  ;  for  laboratory 
microscope,  61. 

Standard  distance   (250  mm.)  at  which 
the  virtual  image  is  measured,  97. 

Starch,  examination  of,  90. 

Stokes  and  Angstrom's  law  of  absorption 
spectra,  123. 

Storing  preparations,  171. 

Substage,  67. 

Substances  for  crystallography,  1  Si. 

Sul phonal  with  polarizer,  139. 

Sulphuric  ether,  177. 

Swaying  of  image,  46. 

System,    back,    front,    intermediate,    of 
lenses,  10. 


Table,  for  immersion  fluid,  213  ;  of  mag- 
nification and  valuation  of  ocular 
micrometer,  99  ;  of  tube-length  and 
thickness  of  cover-glass* s,  15  ; 
natural  sines,  third  page  of  cover  ; 
of  weights  and  measures,  second 
page  of  cover  ;  of  numerical  aper- 
ture, 20  ;  record  ,  for  photo-microg- 
raphy, 204  ;  size  of  fields,  28  ;  of  val- 
uations of  ocular  micrometer,  99. 

Temporary  mounting,  146. 

Terminology  of  objectives,  11. 

Test  of  chromatic  and  spherical  aberra- 
tion, 210. 

Tester,  cover-glass,  144,  145  ;  for  homo- 
geneous liquids,  55. 

Testing  a  camera,  185;  ink.  216;  a  mi- 
croscope and  its  parts,  60. 

Test-plate,  Abbe's,  method  of  using,  210. 

Textile  fibers,  examination  of,  90. 

Thickness  of  cover  glass  for  non-adjust- 
able objectives,  table,  15  ;  of  serial 
sections,  170. 

Tissues,  arranging  for  sections,  168  ;  fix- 
ing or  hardening,  157,  164. 

Tolles-Mayall  mechanical  stage,  65. 

Transections,  169. 

Transfering  drawings,  217  ;  sections,  160. 

Transmitted  light,  35. 

Transparent  objects  having  curved  out- 
lines, relative  position  in  microscop- 
ic preparations,  83. 

Triplet,  achromatic,  7. 

Tripod,  7  ;  as  focusing  glass,  19S. 

Tube  of  microscope,  Frontispiece. 

Tube-length,  14-16,  54;  for  cover-glass 
adjustment,  53,  54;  importance  of, 
53.  54;  microscopical,  14,  15;  of 
various  opticians,  table,  15  ;  and  op- 
tical combinations,  104. 

Turn-table,  148. 

U— V— W-X 

Unadjustable  objectives,  13. 

Under-correction,  5. 

Unit  of  measures,  in  micrometry,  100  ; 
of  wave  length,    1  29. 

Uranium  nitrate  spectrum  of,  135. 

Usago  paper,  160. 

Valuation  of  ocular  micrometer,  102, 
103  ;  table,  99 

Variable  objective,  13. 

Varying  magnification  of  compound  mi- 
croscope, 97. 

Varying  ocular  micrometer  valuation, 
105. 

Velocity  under  microscope,  SS. 


/A/'/    \ 


Vertical  camera,   188, 

•"7 
Vials  lor  preparation    159 
Virtual  image,  s,  6,  to,  ,^1  ;  standard  «1  i^ - 

tance  at  which  measured,  97. 
Vision,  double  or  binocular. 
Ward's  eye  shade,  5  1. 

Waste  howl.    162. 

Water  immersion  objective,  17,  im,  55; 
light  utilized,  [8;  numerical  aper 
ture,  20 

Water,  for  immersion  objectives,  55  ;  re- 
moval, 58;  soliil  sediment  in,  r.80. 


Wave 

ot.    : 

Weights  and  measun 

Wo) laston's  camera  lucida,  95,  1 1 1. 

Work  room  for  photO-111 

Work  table,  ]>osii  ion,  < 

Working  distance  <>i  in 

jective,  1 1 ,  33  ;  determination  of 

oculai  s, 
Writing  diamond,  1 7  j. 

X\  lciie.  159,  1 76  ;  balsam,  1 
Xylol,  German  form  ofxylet 


nOmn  L1BKARY 

If-  C.  State  College 


TABLE  OF  NATURAL  SIN] 
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Minutes 


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